[Clinical and biological features of patients with essential thrombocythaemia according to their mutational status JAK2 or CALR: Single-center study of 40 patients and review of the literature]. / Caractéristiques clinico-biologiques des patients avec thrombocytémie essentielle en fonction de leur statut mutationnel JAK2 ou CALR : étude monocentrique de 40 patients et revue de la littérature.

BACKGROUND: Somatic mutations in the calreticulin gene (CALR) were recently described in essential thrombocythemia (ET) and primary myelofibrosis with non-mutated JAK2 or MPL. The aim of this single-center study was to compare the clinical and biological features of ET patients according to their mutational status. METHODS: We included 40 patients with ET followed in hematology consultation. The JAK2 V617F mutation was assessed by quantitative PCR. For the detection of CALR mutations, we performed a PCR amplification of CALR exon 9 followed by direct sequencing. RESULTS: Among 40 study patients, 23 (57.5%) harbored V617F JAK2, 12 of the 17 patients without JAK2 mutation harbored CALR, no patient expressed MPL mutation and 5 were negative for all three mutations. Five types of mutations were identified with predominance of 52bp deletion and 5bp insertion (7/12 and 2/12 respectively). The incidence of thrombotic events at diagnosis was significantly higher in JAK2 mutated patients (P<0.05). Biologically, patients with CALR mutation had significantly higher platelet count (P<0.01) and significantly lower hemoglobin level (P<0.05) than those with V617F JAK2 mutation. CONCLUSION: JAK2 and CALR mutation screening in ET has a diagnostic value. Each mutation displays a distinct phenotype with uncertain impact on long-term outcome.

[Clozapine rechallenge in resistant schizophrenia disorder affecting "super sensitive" patients, after neutropenia under clozapine: a case report]. / Réintroduction de la clozapine chez les patients souffrant de schizophrénie résistante "super répondeurs", après neutropénie sous clozapine: à propos d'un cas.

INTRODUCTION AND OBJECTIVE: The frequency of agranulocytosis induced by psychoactive drugs is estimated the first year of around 0.8% under clozapine, against 0.13% under chlorpromazine (King and Wager, 1998 [3]). It is associated with a mortality rate of 5 to 10%, and requires heavy treatment, usually in an intensive care unit. The objective of this paper is to present a practical therapeutic answer (clozapine rechallenge with filgrastim) through a case report following a neutropenia episode preventing clozapine use. CASE AND METHODS: B.N. aged 35, native of Martinique, shows a resistant schizophrenia disorder "ultra sensitive" to clozapine. Without any treatment, after 4 years in stable clinical state under clozapine, B.N. suffered three neutropenia episodes when absorbing clozapine (2008, 2010 and 2011). First, a literature survey was conducted along with a consultation of the head of pharmacovigilance regional center and the hematology referee. Then, a 4th clozapine treatment was decided under cover of filgrastim (G-CSF), the role of which is to limit the risk of a new neutropenia. After stopping all psychoactive drugs, except morphine, the subject benefited from a first 0.3mg filgrastim injection, the day before re-introducing 25mg clozapine. Before treatment: Leucocytes=4.8 G/L while absolute neutrophils count=2.4 G/L. Filgrastim injections were carried out at a rate of two 0.3mg injections per week. Clozapine was increased to reach 25mg every 3 days and electroconvulsivotherapy continued fortnightly while supervision was double: on the first hand, daily and clinical search for an increase in body temperature and signs of treatment intolerance, and on the other hand biological surveillance with NFS three times a week besides weekly clozapinemia. The well-informed consent of the patient was obtained. RESULTS: Signs of improvement were noticed as early as the 8th day and after 8 weeks of treatment and 31 sessions of ECT, the patient was stabilized under clozapine at 300 mg per day. The evolution is clearly favorable, as PANNS evolved from 158 to 90. Neutropenia episodes were not observed with a lowest measured rate of 1.9 G/L neutrophils. The filgrastim dosage was then reduced to 0.3mg per week from the 7th week onwards, along with the pursuit of a weekly NFS supervision throughout the treatment. Tolerance is satisfying, with an improvement in lipid check, glycaemia, blood pressure and QT intervals during ECG. DISCUSSION AND CONCLUSION: The B.N. case isn't an isolated one as several articles refer to filgrastim use, combined with clozapine. This confirms the role of hematopoietic cytokines (mainly G-CSF) in neutropenia episodes induced by clozapine. Filgrastim dosage appears to be an important point with regards to the risk of a new neutropenia episode. Let's mention also that it is not a harmless treatment, it could hide the occurrence of neutropenia, besides it's expensive and invasive. Clinical and biological supervision is essential as the probability of an enhanced malignant hemopathy is low but nonetheless present. We also noticed a "biased notoriety of the clozapine", with the association with other hematotoxic molecules, the existence of a circadian rhythm of neutrophils or G-CSF, along with transitional or ethnical neutropenia. These points should be discussed thoroughly before exclusively accusing clozapine; this in turn would have consequences regarding the possibility of treatment resumption. Finally, association with lithium is also an option; several cases have already been reported.

[Human immunodeficiency virus and venous thromboembolism: Role of direct oral anticoagulants]. / Modalités du traitement de la maladie veineuse thromboembolique du patient VIH par les anticoagulants oraux directs.

Nowadays, thanks to highly active antiretroviral therapy (HAART), human immunodeficiency virus (HIV) infection is transforming into a chronic disease. The life expectancy of people living with HIV (PWH) has increased, as well as their risk of developing several co-morbidities, in particular cardiovascular diseases. In addition, the incidence of venous thromboembolism (VTE) is increased in PWH with a 2 to 10 times higher incidence when compared to the general population. Over the last decade, direct oral anticoagulants (DOACs) have been widely used in the treatment and prevention of VTE and non-valvular atrial fibrillation. DOACs are characterized by a rapid onset of activity, a predictable response and a relatively wide therapeutic window. Nevertheless, drug interactions exist between HAART and DOACs, exposing PWH to a theoretically increased bleeding or thrombotic risk. DOACs are substrates of the transport protein P-glycoprotein and/or of isoforms of cytochromes P450 pathway, which can be affected by some antiretroviral drugs. Limited guidelines are available to assist physicians with the complexity of those drug-drug interactions. The aim of this paper is to provide an updated review on the evidence of the high risk of VTE in PWH and the place of DOAC therapy in this population.

French National Diagnostic and Care Protocol for antiphospholipid syndrome in adults and children.

Antiphospholipid syndrome (APS) is a chronic autoimmune disease involving vascular thrombosis and/or obstetric morbidity and persistent antibodies to phospholipids or certain phospholipid-associated proteins. It is a rare condition in adults and even rarer in children. The diagnosis of APS can be facilitated by the use of classification criteria based on a combination of clinical and biological features. APS may be rapidly progressive with multiple, often synchronous thromboses, resulting in life-threatening multiple organ failure. This form is known as "catastrophic antiphospholipid syndrome" (CAPS). It may be primary or associated with systemic lupus erythematosus (associated APS) and in very rare cases with other systemic autoimmune diseases. General practitioners and paediatricians may encounter APS in patients with one or more vascular thromboses. Because APS is so rare and difficult to diagnosis (risk of overdiagnosis) any suspected case should be confirmed rapidly and sometimes urgently by an APS specialist. First-line treatment of thrombotic events in APS includes heparin followed by long-term anticoagulation with a VKA, usually warfarin. Except in the specific case of stroke, anticoagulants should be started as early as possible. Any temporary discontinuation of anticoagulants is associated with a high risk of thrombosis in APS. A reference/competence centre specialised in autoimmune diseases must be urgently consulted for the therapeutic management of CAPS.

[Contribution of antiphosphatidylserine/prothrombin (anti-PS/PT) antibody detection in the diagnosis and management of antiphospholipid syndrome (APS)]. / Apport de la détection des anticorps antiphosphatidylsérine/prothrombine (aPS/PT) dans le diagnostic et la prise en charge du syndrome des antiphospholipides (SAPL).

Antiphospholipid syndrome (APS) is an autoimmune disease and one of the most common causes of acquired thrombophilia. It is characterised by the occurrence of thrombotic or obstetric events associated with the presence of persistent antiphospholipid antibodies. The diagnosis can be challenging, particularly because some biological tests can be disturbed by anticoagulant treatment or inflammation. In the recent years, new antiphospholipid antibodies, including anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT), have emerged but their clinical significance and causality remain uncertain. Biologically, several studies have found a strong correlation between the presence of lupus anticoagulant (LA) and anti-PS/PT antibodies. Clinically, the presence of anti-PS/PT antibodies is associated with an increased risk of thrombosis and obstetric complications. There is also an association with thrombocytopenia, suggesting that the presence of anti-PS/PT antibodies may be associated with more severe clinical APS. Among seronegative APS patients, 6-17% of patients are positive for anti-PS/PT antibodies. This might influence the therapeutic management of patients. This article aims to provide an update on contribution of anti-PS/PT antibodies detection for the diagnosis and management of APS.

Arterial and venous thrombosis is associated with different angiogenic cytokine patterns in patients with antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is defined as a combination of antiphospholipid antibodies, arterial and/or venous thrombosis, and, in women, recurrent fetal loss. The mechanisms underlying this prothrombotic tendency are unclear. Here we determined plasma levels of the angiogenic growth factors vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and stromal cell-derived factor-1 (SDF-1) in 34 patients with APS (median age 40 years) compared with 180 healthy controls and with 80 age-matched deep-venous thrombosis patients in whom the diagnosis of APS had been excluded. All of the patients met updated APS criteria and two-thirds of them were triply positive for antiphospholipid antibodies (lupus anticoagulant, anti-beta2-glycoprotein I and anti-cardiolipin antibodies). Angiogenic cytokines were quantified at least 6 months after an acute thrombotic event. VEGF levels were similar in the patients and controls, but were significantly higher in the patients with arterial thrombosis than in the patients with venous thrombosis. Plasma levels of SDF-1 and PlGF were significantly elevated in the patients, regardless of the arterial/venous nature of the thrombosis. Together, these results suggest that APS is associated with an angiogenic process, but that the angiogenic signal differs between patients with arterial and venous thrombosis. Lupus (2010) 19, 837-843.

[Acquired hemophilia as the presenting manifestation of neoplasia: diagnostic workup and monitoring]. / Hémophilie A acquise précédant l'apparition d'un cancer: quelles explorations et quelle surveillance?

Acquired haemophilia is a rare disorder caused by the development of autoantibody to factor VIII. It is sometimes associated with malignancies, and usually appears during disease course. In rare instances, acquired haemophilia is the presenting manifestation of a malignant disease. We report a 76-year-old man, who presented with spontaneous haematomas of his four limbs. A factor VIII inhibitor was found and the patient diagnosed with acquired haemophilia. Initial etiologic diagnostic workup including a thoracic and abdominal computed tomographic scan was negative. Factor VIII inhibitor disappeared on corticosteroids and factor VIII level normalized. Seven months later, the patient died from a multimetastatic cancer. About 15% of acquired haemophilia are associated with malignant disease (malignant lymphoma or solid neoplasia). Although rare, the development of a factor VIII inhibitor few months before the diagnosis of the malignant disease raised the issue of the appropriate initial investigations and further monitoring to recommend these patients. We propose a regular clinical monitoring and a thoracic and abdominal computed tomographic scan at six-month follow-up to screen for malignant disease.

[Review: delta-storage pool disease]. / Mise au point : la maladie du pool vide plaquettaire.

Platelet storage pool disease is a qualitative platelet disorder associated with variable degrees of reduction in the numbers and contents of dense granules (delta-granules). Electron microscopy is the major tool for biological diagnosis. Patients presenting with this platelet disorder generally show a mild bleeding syndrome.

[Laboratory diagnosis of antiphospholipid syndrome: From criteria to practice]. / Diagnostic biologique du syndrome des antiphospholipides : des critères à la pratique.

The antiphospholipid syndrome is a bioclinical entity defined by thrombosis and/or obstetrical complications in the presence, at least 12 weeks apart, of antiphospholipid antibodies detected by coagulation test (lupus anticoagulant) or immunological assays (anticardiolipin, anti-ß2-glycoprotein I antibodies). Biological markers' improvement such as anti-phosphatidylserine/prothrombin and biological score should allow better patients' management and preventive therapeutic for thrombosis and obstetrical complications. This review describes different types of antibodies, link between biological profile and risk level of thrombosis events/obstetrical complications and gives practical advice to interpret biological results.

[Gardner-Diamond syndrome in a young man: A case report and literature review]. / Syndrome de Gardner-Diamond : à propos d'un cas chez un homme jeune et revue de la littérature.

INTRODUCTION: Gardner-Diamond syndrome is a rare condition secondary to a sensitization to self-erythrocytes. It is predominantly seen in women and presents as a painful ecchymotic disorder. An underlying psychiatric disease or a triggering psychological stress is of important diagnostic value. CASE REPORT: We report a 24-year-old patient who presented with intermittent spontaneous painful ecchymosis since 5 years. Complementary investigations failed to identify an organic disorder. Gardner-Diamond syndrome was retained because of the clinical presentation, the negativity of diagnostic work-up and the identification of a psychological trauma. Patient management (pain, psychological support) is difficult, justifying a multidisciplinary approach. CONCLUSION: Gardner-Diamond syndrome is a rare and unrecognized disorder, which should be discussed in the presence of ecchymotic or purpuric lesions that do not have a diagnostic orientation. Early recognition of this disorder enables initiation of an appropriate management, but also limits unnecessary additional explorations.

[Acquired mutation of JAK2 tyrosine kinase and polycythaemia vera]. / Mutation acquise de la tyrosine kinase Jak2 et maladie de Vaquez.

Polycythaemia vera is an acquired myeloproliferative disorder characterised by a polycythaemia resulting of a clonal disorder arising in a multipotent hematopoietic stem cell. The increase of red cell mass exposes to a high risk of arterial or venous thrombosis and thus requires a cytoreductive treatment. An acquired genetic mutation in exon 12 of the JAK2 tyrosine kinase gene, leading to a substitution of a valine to a phenylalanine (V617F), has been described in most polycythaemia vera patients. This mutation increases the phosphorylation activity of JAK2, promotes the spontaneous cellular growth and induces erythrocytosis in a mouse model. Prevalence studies of V617F JAK2 mutation in different myeloproliferative disorders have found this genetic alteration in half of idiopathic myelofibrosis and in one third of essential thrombocythaemia. This finding is a huge progress in the understanding of polycythaemia vera physiopathology, it will be also an useful tool for the diagnosis of myeloproliferative disorders and it opens a new field for the development of targeted therapeutic approaches in these disorders.

[Acute megakaryoblastic leukemia (AML-7) in a woman of 95 years]. / Leucémie aiguë mégacaryoblastique (LAM-7) chez une patiente de 95 ans.

Acute leukemia of megakaryocyte lineage (AML-7) is a rare entity defined by a blastic proliferation of which a part (>or= 50%) is represented by megakaryoblasts. We report the case of a 95 year old woman presenting a AML-7 secondary to a myelodysplastic syndrome (MDS), that represents an unusual form of MDS acutisation.

[Laboratory diagnosis of antiphospholipid syndrome]. / Diagnostic biologique du syndrome des antiphospholipides.

PURPOSE: The antiphospholipid syndrome is a bioclinical entity defined by the occurrence of thromboses, and/or obstetrical complications in the persistent presence of antiphospholipid antibodies, i.e. lupus anticoagulant and/or anticardiolipin antibodies. This review focuses on the methods for antiphospholipid antibodies detection and their clinical usefulness. CURRENT KNOWLEDGE AND KEY POINTS: Lupus anticoagulant is the strongest risk factor for thrombosis in antiphospholipid syndrome. Twenty years after its description, anticardiolipin ELISA, despite a still improvable standardization and its lack of specificity, is still required for sensitive diagnosis of antiphospholipid syndrome. FUTURE PROSPECTS AND PROJECTS: A better knowledge of the beta-2-glycoprotein-I role in the pathophysiology of antiphospholipid syndrome might lead to the development of new markers of thrombotic risk.

[Prognostic factors in chronic lymphocytic leukaemia: contribution of recent biological markers]. / Facteurs pronostiques de la leucémie lymphoïde chronique: apport des marqueurs biologiques récents.

Chronic lymphocytic leukemia (CLL) is the most common lymphoid hemopathy in elderly. Diagnosis of CLL is easily made with a full blood count and immunophenotyping, but there is an heterogeneity in clinical evolution. Until now, scheduling of treatment is based on Rai or Binet staging systems. These staging systems can not distinguish patients with a rapid evolution and thus who will need an earlier treatment. In order to detect these patients, it is useful to have some relevant markers to predict disease evolution. This article reviews recent biologic markers that can be used to evaluate long term prognosis of CLL patients.

Spontaneous soft tissue hematomas.

Spontaneous muscle hematomas are a common and serious complication of anticoagulant treatment. The incidence of this event has increased along with the rise in the number of patients receiving anticoagulants. Radiological management is both diagnostic and interventional. Computed tomography angiography (CTA) is the main tool for the detection of hemorrhage to obtain a positive, topographic diagnosis and determine the severity. Detection of an active leak of contrast material during the arterial or venous phase is an indication for the use of arterial embolization. In addition, the interventional radiological procedure can be planned with CTA. Arterial embolization of the pedicles that are the source of the bleeding is an effective technique. The rate of technical and clinical success is 90% and 86%, respectively.

Increased Performances of the Biological Diagnosis of the Antiphospholipid Syndrome by the Use of a Multiplex Assay.

Antiphospholipid syndrome (APS) is characterized by development of venous and/or arterial thrombosis and pregnancy morbidity. Biological criteria are the persistent presence of lupus anticoagulant (LA) and/or anti-cardiolipin (aCL) and/or anti-B2GP1 autoantibodies' positivity. The assays' performances are of crucial importance. We evaluated a multiplex assay allowing simultaneous detection of IgG anti-cardiolipin, anti-B2GP1, and anti-factor II. 300 samples were tested. Patients were categorized according to clinical scores of APS from 0 to 3 based on presence or not of arterial or venous thrombosis, fetal loss, and autoimmunity. We used a multiplex assay for APS for simultaneous detection of aCL, anti-B2GP1, and factor II and compared its performances to ELISA assays. Presence of LA was also assessed. We performed a correlation study of the tested assays and compared their clinical efficacy by ROC curve analysis. We obtained significantly higher performances with the multiplex assay than ELISA with higher area under the curve (AUC). The disease rate increased with the number of positive markers from 9% for 1 marker to 100% for 4 markers positive for patients with high risk scores. The multiplex APS assay exhibited higher performances particularly in case of primary APS and is useful for rapid diagnosis of APS.

Oxidation of beta2-glycoprotein I (beta2GPI) by the hydroxyl radical alters phospholipid binding and modulates recognition by anti-beta2GPI autoantibodies.

We investigated whether beta2-glycoprotein I (beta2GPI), the key antigen in the antiphospholipid syndrome, is susceptible to oxidative modifications by the hydroxyl radical (*OH) that may influence its lipid-binding and antigenic properties. The effects on human and bovine beta2GPI of *OH free radicals generated by gamma-radiolysis of water with 137Cs were studied. Radiolytic *OH caused a dose-dependent loss of tryptophan, production of dityrosine and carbonyl groups. dimerization and/or extensive aggregation of beta2GPI. It ensued a reduction in affinity binding to cardiolipin liposomes and loss of beta2GPI-dependent autoantibody binding to immobilized cardiolipin. Patient anti-beta2GPI antibodies (n = 20) segregated into two groups based on the effect in the beta2GPI-ELISA of beta2GPI pretreatment with *OH: enhancement (group A, n = 10) or suppression (group B, n = 10) of IgG binding. The avidities of group A antibodies for fluid-phase beta2GPI were low but increased in a dose-dependent manner upon beta2GPI irradiation, in relation to protein crosslinking. Distinguishing features of group B antibodies included higher avidities for fluid-phase beta2GPI that was no longer recognized after *OH treatment, and negative anticardiolipin tests suggesting epitope location near the phospholipid binding site. The *OH scavengers thiourea and mannitol efficiently protected against all above changes. Therefore, oxidative modifications of beta2GPI via *OH attack of susceptible amino acids alter phospholipid binding, and modulate recognition by autoantibodies depending on their epitope specificities. These findings may be of clinical relevance for the generation and/or reactivity of anti-beta2GPI antibodies.

Species specificity of anti-beta 2 glycoprotein I autoantibodies and its relevance to anticardiolipin antibody quantitation.

Some patients suspected of having antiphospholipid antibody syndrome (APS) were found to be positive for anti-beta 2 glycoprotein I (beta 2GPI) antibodies despite negative results for antibodies to cardiolipin (ACA). Since the major source of beta 2GPI in the ACA assay is animal (usually bovine) serum, we studied the influence on ACA quantitation of the species specificity of anti-beta 2GPI antibodies from patients with various autoimmune disorders, mostly systemic lupus erythematosus and primary APS. Ninety-seven sera were selected based on IgG (n = 76) or IgM (n = 64) positivity by ELISA using gamma-irradiated plates coated with human or bovine purified beta 2GPI. A higher proportion of IgM (43.7%) than IgG (7.9%) reacted to human, but not bovine, beta 2GPI. Furthermore, from the samples reactive to both proteins, the ratio of antibody level against bovine to that against human beta 2GPI was 1.08 +/- 0.58 for IgG and 0.58 +/- 0.3 for IgM (p < 10(-5)). IgG and IgM ACA were detected in 78 and 40 sera, respectively; concordance between the two ELISAs for ACA and anti-beta 2GPI antibodies was 94% for IgG and 75% for IgM. Out of 28 IgM showing recognition restricted to human beta 2GPI, 21 were missed by the ACA assay, possibly because of lower concentrations of beta 2GPI in those patients' sera. The antibody reactivity pattern towards human and bovine beta 2GPI of individual sera showed no variation with time and was related to the relative antibody avidity for each protein. A murine anti-human beta 2GPI monoclonal antibody, 9G1, that cross-reacts with bovine beta 2GPI, competed to a large extent with the patients' anti-beta 2GPI antibody binding sites whatever isotype involved or protein recognized. Therefore, anti-beta 2GPI antibodies of IgM isotype display a marked preference for human compared to bovine beta 2GPI responsible for frequent inconsistencies in the ACA assay.

Heterogeneity and immunochemical properties of anti-beta2-glycoprotein I autoantibodies.

Most anticardiolipin antibodies (ACA) associated with antiphospholipid syndrome (APS) are directed against epitopes expressed on beta2-glycoprotein I (beta2GPI). Despite a good correlation between standard ACA assays and those using purified human beta2GPI as the sole antigen, some sera from APS patients only react in the latter. This is indicative of heterogeneity in anti-beta2GPI antibodies. To characterize their reactivity profiles, human and bovine beta2GPI were immobilized on gamma-irradiated plates (beta2GPI-ELISA), plain polystyrene precoated with increasing cardiolipin concentrations (CL/beta2GPI-ELISA), and affinity columns. Fluid-phase inhibition experiments were also carried out with both proteins. Of 56 selected sera, restricted recognition of bovine or human beta2GPI occurred respectively in 10/29 IgA-positive and 9/22 IgM-positive samples, and most of the latter (8/9) were missed by the standard ACA assay, as expected from a previous study. Based on species specificity and ACA results, IgG-positive samples (53/56) were categorized into three groups: antibodies reactive to bovine beta2GPI only (group I) or to bovine and human beta2GPI, group II being ACA-negative, and group III being ACA-positive. The most important group, group III (n = 33) was characterized by (i) binding when beta2GPI was immobilized on gamma-irradiated polystyrene or cardiolipin at sufficient concentration (regardless of beta2GPI density, as assessed using 125I-beta2GPI); (ii) and low avidity binding to fluid-phase beta2GPI (Kd in the range 10(-5) M). In contrast, all six group II samples showed (i) ability to bind human and bovine beta2GPI immobilized on non-irradiated plates; (ii) concentration-dependent blockade of binding by cardiolipin, suggesting epitope location in the vicinity of the phospholipid binding site on native beta2GPI; (iii) and relative avidities approximately 100-fold higher than in group III. Group I patients were heterogeneous with respect to CL/beta2GPI-ELISA and ACA results (6/14 scored negative), possibly reflecting antibody differences in terms of avidity and epitope specificity. Affinity fractionation of 23 sera showed the existence, in individual patients, of various combinations of antibody subsets solely reactive to human or bovine beta2GPI, together with cross-species reactive subsets present in all samples with dual reactivity namely groups III and II, although the latter antibodies were poorly purified on either column. Therefore, the mode of presentation of beta2GPI greatly influences its recognition by anti-beta2GPI antibodies with marked inter-individual heterogeneity, in relation to ACA quantitation and, possibly, disease presentation and pathogenesis.