Role of the mechanotransductor PIEZO1 in megakaryocyte differentiation.

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34+ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 µM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41+CD42+ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.

Quantitative determination of plasma free and total concentrations of antivitamin K drugs using a new sensitive and rapid LC-MS/MS method: Application in hemodialysis patients.

BACKGROUND AND AIMS: Vitamin K antagonists (VKAs) are the first-line anticoagulants used in end stage renal disease. This population experiences a significant variability in their International Normalized Ratio (INR) over time. There is a need for methods allowing the study of the pharmacokinetics of free and total concentrations of VKAs to explain INR variability. MATERIALS AND METHODS: We developed and validated a high-performance liquid chromatography-tandem mass spectrometry method allowing the quantification of warfarin and fluindione free and total plasma concentrations. Chromatographic separation was achieved in a raptor biphenyl column and the spectrometry acquisition was set in multiple reaction monitoring mode after negative electrospray ionization. We then applied it in describing the plasma free and total concentrations of VKAs in samples from 50 hemodialysis patients. RESULTS: The developed method is rapid, sensitive and specific. Our cohort results showed a correlation between free and total VKA concentrations. The free VKA concentrations tended to be higher in patients with higher INR. Although VKAs are highly albumin-bound drugs, albumin concentration did not totally explain the high inter-individual total VKA concentrations variability. CONCLUSION: This opens the door to further studies to understand the factors involved in their variability.

Combining imaging flow cytometry and machine learning for high-throughput schistocyte quantification: A SVM classifier development and external validation cohort.

BACKGROUND: Schistocyte counts are a cornerstone of the diagnosis of thrombotic microangiopathy syndrome (TMA). Their manual quantification is complex and alternative automated methods suffer from pitfalls that limit their use. We report a method combining imaging flow cytometry (IFC) and artificial intelligence for the direct label-free and operator-independent quantification of schistocytes in whole blood. METHODS: We used 135,045 IFC images from blood acquisition among 14 patients to extract 188 features with IDEAS® software and 128 features from a convolutional neural network (CNN) with Keras framework in order to train a support vector machine (SVM) blood elements' classifier used for schistocytes quantification. FINDING: Keras features showed better accuracy (94.03%, CI: 93.75-94.31%) than ideas features (91.54%, CI: 91.21-91.87%) in recognising whole-blood elements, and together they showed the best accuracy (95.64%, CI: 95.39-95.88%). We obtained an excellent correlation (0.93, CI: 0.90-0.96) between three haematologists and our method on a cohort of 102 patient samples. All patients with schistocytosis (>1% schistocytes) were detected with excellent specificity (91.3%, CI: 82.0-96.7%) and sensitivity (100%, CI: 89.4-100.0%). We confirmed these results with a similar specificity (91.1%, CI: 78.8-97.5%) and sensitivity (100%, CI: 88.1-100.0%) on a validation cohort (n=74) analysed in an independent healthcare centre. Simultaneous analysis of 16 samples in both study centres showed a very good correlation between the 2 imaging flow cytometers (Y=1.001x). INTERPRETATION: We demonstrate that IFC can represent a reliable tool for operator-independent schistocyte quantification with no pre-analytical processing which is of most importance in emergency situations such as TMA. FUNDING: None.

[Chronic lymphocytic leukemia with t(14 ;18) and trisomy 12: a case report]. / Leucémie lymphoïde chronique avec t(14;18) et trisomie 12 : un cas clinique.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm defined by the presence of at least 5×109 G/L monoclonal B lymphocytes in the peripheral blood. It is the most common type of leukemia in adult patients from Western countries. CLL is characterized by a gradual accumulation of small, longliving, immunologically dysfunctional, morphologically mature-appearing B-lymphocytes in blood, bone marrow and lymphoid tissues. It has also been reported that CLL cells have a proliferation rate higher than previously recognized, particularly in the lymphoid tissues. The flow cytometry analysis of typical CLL identifies a monotypic B-cell population expressing a low level of surface immunoglobulins, light chain being either kappa or lambda-, CD5+, CD19+, CD23+, CD79b (dim), negative for FMC7 and CD10. Clinical presentation, course and outcome are highly variable. Interphase fluorescent in situ hybridization (I-FISH) identifies chromosomal abnormalities in about 80% of cases, most commonly involving 13q14 (55%), 11q22-23 (18%), or 17p13 deletions (7%) and trisomy 12 (16%). Therefore, five prognostic categories have been defined with a statistical model, showing the shortest median survival and treatment-free intervals in patients harboring 17p and 11q deletions, followed by trisomy 12 and a normal karyotype, whereas 13q deletion as the sole abnormality is associated with the best prognosis. We report here a rare case of CLL in a 54 year-old-man.

[Paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome: a case report]. / Hémoglobinurie paroxystique nocturne et syndrome myélodysplasique : à propos d'un cas.

Paroxysmal nocturnal hemoglobinuria is a rare clonal non-malignant disease, linked to an acquired PIG-A gene mutation. We report the case of 81 years old patient hospitalized for articular ache, swelling and temporal arteries' induration in which we diagnose PNH associated with refractory cytopenia with multilineage dysplasia.

Thrombin time and anti-IIa dabigatran's activity: hypothesis of thrombin time's predictive value.

Dabigatran etexilate (Pradaxa®) is a new oral anticoagulant, competitive inhibitor, selective, fast, direct and reversible of thrombin. Dabigatran has an impact on a large panel of used coagulation tests. There is no relationship between thrombin time's lengthening and anti-IIa activity. This study defines thrombin time's predictive value, when its time is normal. The result of negative value is 97,6%. 255 patients were studied between January 2013 and July 2014. Thrombin time and anti-IIa activity were dosed for each patient. This study can be an assistant for therapeutic decision for laboratories without specialized test.

[Multiorgan failure after sickle cell vaso occlusive attack: integrated clinical and biological emergency]. / Complications en cascade avec défaillance multiviscérale au cours d'une drépanocytose : gestion coordonnée de l'urgence au laboratoire et au service de réanimation médicale.

We describe the case of a 30-year-old patient, suffering from composite S/ß + sickle cell disease. He was hospitalized following a vaso-occlusive attack with acute bone pains. Despite an analgesic treatment and transfusion of three units of red blood cells, a non-regenerative anemia appeared within 24 hours. One day later an acute chest syndrome with atelectasis of the left lung and desaturation and multi-organ failure occurred and necessitated the patient's intubation and required him to be placed in an artificial coma. A bronchoalveolar lavage was performed, which eliminated pneumonia but proved, after staining with oil red O, many neutral fatty acid microvacuoles in more than 80% of macrophages, suggesting a pulmonary fat embolism. The hypothesis of a bone marrow necrosis causing a pulmonary fat embolism was discussed and confirmed the next day by the characteristic appearance of the bone marrow. A therapeutic protocol associating iteratively bleeding and red blood cells transfusion was administered on the second day with the objective of maintaining haemoglobin S at less than 20% rate. Successive haemoglobin S assay was applied using a high performance liquid chromatography (HPLC) technique with a quick response within one hour after transfusion or bleeding. This protocol resulted in an improvement in the patient's condition, with a gradual normalization of vital signs and extubation twelve days later and discharge without sequelae twenty-five days later. The succession of rare but serious sickle cell complications anaemia which occurred in this patient could be controlled by adapting the laboratory for the clinical emergency.