Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion.

Platelet caspase-1 and Bruton tyrosine kinase activation in patients with COVID-19 is associated with disease severity and reversed in vitro by ibrutinib.

Background: Severity of coronavirus disease 2019 (COVID-19) is often associated with thrombotic complications and cytokine storm leading to intensive are unit (ICU) admission. Platelets are known to be responsible for abnormal hemostasis parameters (thrombocytopenia, raised D-dimers, and prolonged prothrombin time) in other viral infections through the activation of the nucleotide-binding domain leucine repeat rich containing protein 3 inflammasome induced by signaling pathways driven by Bruton tyrosine kinase (BTK) and leading to caspase-1 activation. Objectives: We hypothesized that caspase-1 activation and the phosphorylation of BTK could be associated with the severity of the disease and that ibrutinib, a BTK inhibitor, could inhibit platelet activation. Methods and Results: We studied caspase-1 activation by flow cytometry and the phosphorylation of BTK by Western blot in a cohort of 51 Afro-Carribean patients with COVID-19 disease (19 not treated in ICU and 32 treated in ICU). Patients with a platelet count of 286.7 × 109/L (69-642 × 109/L) were treated by steroids and heparin preventive anticoagulation. Caspase-1 and BTK activation were associated with the severity of the disease and with the procoagulant state of the patients. Furthermore, we showed in vitro that the plasma of ICU patients with COVID-19 was able to increase CD62P expression and caspase-1 activity of healthy platelets and that ibrutinib could prevent it. Conclusions: Our results show that caspase-1 and BTK activation are related to disease severity and suggest the therapeutic hope raised by ibrutinib in the treatment of COVID-19 by reducing the procoagulant state of the patients.

Perls' Stain Guidelines from the French-Speaking Cellular Hematology Group (GFHC).

In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies.

The CADM1 tumor suppressor gene is a major candidate gene in MDS with deletion of the long arm of chromosome 11.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis leading to peripheral cytopenias and in a substantial proportion of cases to acute myeloid leukemia. The deletion of the long arm of chromosome 11, del(11q), is a rare but recurrent clonal event in MDS. Here, we detail the largest series of 113 cases of MDS and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) harboring a del(11q) analyzed at clinical, cytological, cytogenetic, and molecular levels. Female predominance, a survival prognosis similar to other MDS, a low monocyte count, and dysmegakaryopoiesis were the specific clinical and cytological features of del(11q) MDS. In most cases, del(11q) was isolated, primary and interstitial encompassing the 11q22-23 region containing ATM, KMT2A, and CBL genes. The common deleted region at 11q23.2 is centered on an intergenic region between CADM1 (also known as Tumor Suppressor in Lung Cancer 1) and NXPE2. CADM1 was expressed in all myeloid cells analyzed in contrast to NXPE2. At the functional level, the deletion of Cadm1 in murine Lineage-Sca1+Kit+ cells modifies the lymphoid-to-myeloid ratio in bone marrow, although not altering their multilineage hematopoietic reconstitution potential after syngenic transplantation. Together with the frequent simultaneous deletions of KMT2A, ATM, and CBL and mutations of ASXL1, SF3B1, and CBL, we show that CADM1 may be important in the physiopathology of the del(11q) MDS, extending its role as tumor-suppressor gene from solid tumors to hematopoietic malignancies.

Plasma microparticles of intubated COVID-19 patients cause endothelial cell death, neutrophil adhesion and netosis, in a phosphatidylserine-dependent manner.

COVID-19 has compelled scientists to better describe its pathophysiology to find new therapeutic approaches. While risk factors, such as older age, obesity, and diabetes mellitus, suggest a central role of endothelial cells (ECs), autopsies have revealed clots in the pulmonary microvasculature that are rich in neutrophils and DNA traps produced by these cells, called neutrophil extracellular traps (NETs.) Submicron extracellular vesicles, called microparticles (MPs), are described in several diseases as being involved in pro-inflammatory pathways. Therefore, in this study, we analyzed three patient groups: one for which intubation was not necessary, an intubated group, and one group after extubation. In the most severe group, the intubated group, platelet-derived MPs and endothelial cell (EC)-derived MPs exhibited increased concentration and size, when compared to uninfected controls. MPs of intubated COVID-19 patients triggered EC death and overexpression of two adhesion molecules: P-selectin and vascular cell adhesion molecule-1 (VCAM-1). Strikingly, neutrophil adhesion and NET production were increased following incubation with these ECs. Importantly, we also found that preincubation of these COVID-19 MPs with the phosphatidylserine capping endogenous protein, annexin A5, abolished cytotoxicity, P-selectin and VCAM-1 induction, all like increases in neutrophil adhesion and NET release. Taken together, our results reveal that MPs play a key role in COVID-19 pathophysiology and point to a potential therapeutic: annexin A5.

Platelet Counting: Ugly Traps and Good Advice. Proposals from the French-Speaking Cellular Hematology Group (GFHC).

Despite the ongoing development of automated hematology analyzers to optimize complete blood count results, platelet count still suffers from pre-analytical or analytical pitfalls, including EDTA-induced pseudothrombocytopenia. Although most of these interferences are widely known, laboratory practices remain highly heterogeneous. In order to harmonize and standardize cellular hematology practices, the French-speaking Cellular Hematology Group (GFHC) wants to focus on interferences that could affect the platelet count and to detail the verification steps with minimal recommendations, taking into account the different technologies employed nowadays. The conclusions of the GFHC presented here met with a "strong professional agreement" and are explained with their rationale to define the course of actions, in case thrombocytopenia or thrombocytosis is detected. They are proposed as minimum recommendations to be used by each specialist in laboratory medicine who remains free to use more restrictive guidelines based on the patient's condition.

[Performance analysis of the « Blast ¼ flag on ADVIA® 2120/2120i - Results of a multicenter study]. / Analyse de performance de l'alarme « Blastes ¼ sur ADVIA® 2120/2120i ­ Résultats d'une analyse multicentrique.

Nowadays, laboratories have more efficient haematology analyzers. These analyzers have to be used in the most efficient and the most adapted way according to the internal organisation of laboratories and prescribers' expectations. The aim of this study was to evaluate the performance of the blast flag on ADVIA 2120/2120i, and to suggest what to do, depending on the flag intensity, to identify positive samples the most surely and the most rapidly as possible. MATERIALS AND METHODS: Seven hospital laboratories were included in this prospective study, 148 144 samples of hospital patients were tested during this 4 months study. RESULTS: Sensitivity and specificity of the blast flag are respectively 89,04% and 98,97%. A good correlation between the flag level and the blast count on blood smear is noticed. CONCLUSION: This study could be helpful for laboratories using ADVIA 2120/2120i, to adapt their procedures, depending on the level of the flag provided by the analyser.

Southeast asian ovalocytosis: the need for a carefull observation of red cell indices and blood smear.

Southeast asian ovalocytosis (SAO) is characterized by macro-ovalocytes and ovalo-stomatocytes on blood smear. SAO is common in Malaisia and Papua-New-Guinea where upwards to 40 per cent of the population is affected in some coastal region. Inherited in an autosomal dominant way, illness results from deletion of codons 400-408 in SLC4A1 gene which encodes for band 3 erythrocyte membrane protein. This deletion is responsible for an unusual erythrocyte stiffness and oval shape of the cells on blood smear. Heterozygous carriers are usually asymptomatic whereas homozygous are not viable without an intensive antenatal care. Here, we describe 4 patients diagnosed incidentally by cytogram appearance of the Advia® 2120i (Siemens) representing hemoglobin concentration according to red blood mean cellular volume (GR/VCH).

Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features.

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25-29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7-9%) and autophagosome-like structures (0-3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.

Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre-leukaemic clone resulting in T2-ALL and AML-M0.

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1R174Q mutation who developed at the age of 42 years a T2-ALL and, 2 years after remission, an AML-M0. Both AML-M0 and T2-ALL blast populations demonstrated a loss of 1p36.32-23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2-ALL or in AML-M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood-derived CD34+ cells 5 years prior to T2-ALL development revealed only the missense TET2P1962T mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2-ALL and AML-M0 clones. This result suggests that TET2P1962T mutation in association with germline RUNX1R174Q mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.

Haematological spectrum and genotype-phenotype correlations in nine unrelated families with RUNX1 mutations from the French network on inherited platelet disorders.

BACKGROUND: Less than 50 patients with FPD/AML (OMIM 601309) have been reported as of today and there may an underestimation. The purpose of this study was to describe the natural history, the haematological features and the genotype-phenotype correlations of this entity in order to, first, screen it better and earlier, before leukaemia occurrence and secondly to optimize appropriate monitoring and treatment, in particular when familial stem cell transplantation is considered. METHODS: We have investigated 41 carriers of RUNX1 alteration belonging to nine unrelated French families with FPD/AML and two syndromic patients, registered in the French network on rare platelet disorders from 2005 to 2015. RESULTS: Five missense, one non-sense, three frameshift mutations and two large deletions involving several genes including RUNX1 were evidenced. The history of familial leukaemia was suggestive of FPD/AML in seven pedigrees, whereas an autosomal dominant pattern of lifelong thrombocytopenia was the clinical presentation of two. Additional syndromic features characterized two large sporadic deletions. Bleeding tendency was mild and thrombocytopenia moderate (>50 x10(9)/L), with normal platelet volume. A functional platelet defect consistent with a δ-granule release defect was found in ten patients regardless of the type of RUNX1 alteration. The incidence of haematological malignancies was higher when the mutated RUNX1 allele was likely to cause a dominant negative effect (19/34) in comparison with loss of function alleles (3/9). A normal platelet count does not rule out the diagnosis of FPD/AML, since the platelet count was found normal for three mutated subjects, a feature that has a direct impact in the search for a related donor in case of allogeneic haematopoietic stem cell transplantation. CONCLUSIONS: Platelet dysfunction suggestive of defective δ-granule release could be of values for the diagnosis of FPD/AML particularly when the clinical presentation is an autosomal dominant thrombocytopenia with normal platelet size in the absence of familial malignancies. The genotype-phenotype correlations might be helpful in genetic counselling and appropriate optimal therapeutic management.

MYH10 protein expression in platelets as a biomarker of RUNX1 and FLI1 alterations.

RUNX1 gene alterations are associated with acquired and inherited hematologic malignancies that include familial platelet disorder/acute myeloid leukemia, primary or secondary acute myeloid leukemia, and chronic myelomonocytic leukemia. Recently, we reported that RUNX1-mediated silencing of nonmuscle myosin heavy chain IIB (MYH10) was required for megakaryocyte ploidization and maturation. Here we demonstrate that runx1 deletion in mice induces the persistence of MYH10 in platelets, and a similar persistence was observed in platelets of patients with constitutional (familial platelet disorder/acute myeloid leukemia) or acquired (chronic myelomonocytic leukemia) RUNX1 mutations. MYH10 was also detected in platelets of patients with the Paris-Trousseau syndrome, a thrombocytopenia related to the deletion of the transcription factor FLI1 that forms a complex with RUNX1 to regulate megakaryopoiesis, whereas MYH10 persistence was not observed in other inherited forms of thrombocytopenia. We propose MYH10 detection as a new and simple tool to identify inherited platelet disorders and myeloid neoplasms with abnormalities in RUNX1 and its associated proteins.

Use of hematopoietic progenitor cell count on the Sysmex XE-2100 for peripheral blood stem cell harvest monitoring.

Successful peripheral blood stem cell (PBSC) collection depends on the timing of apheresis based on CD34+ cell enumeration. Because this analysis is expensive and induces organization difficulties, we evaluated hematopoietic progenitor cell (HPC) quantification on the Sysmex XE-2100 as a surrogate analysis. We tested 157 blood samples for CD34+ cells and HPC counts. We found a good linear correlation between HPC and CD34+ and determined simple rules allowing to use HPC count in daily practice. We set a positive cut-off >30 HPC/mm(3) for allowing PBSC harvest and a negative cut-off at 0 HPC/mm(3) for which collection should be delayed. These two situations accounted for 62% of cases and CD34+ cell count by flow cytometry confirmed HPC result in 95% of cases. Between 0 and 30 HPC/mm3, CD34+ enumeration is required for decision-making. We conclude that HPC count may be a useful surrogate for CD34+ enumeration in PBSC harvest monitoring.