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Electrochemical biosensors, which are fast and reliable analytical tools, have attracted particular attention in recent years for their ability to incorporate biomolecules into nanomaterial-designed electrodes. In this study, an electrochemical biosensor for the detection of hydrogen peroxide (H2O2) was developed using a multi-walled carbon nanotube (EPMWC) platform electrochemically modified with poly [Fe (III)-5-Aphen] and using an oxide-reductase enzyme as recognition agent. Amperometry was applied to obtain the calibration curves for H2O2 at the reduction process. The analytical parameters such as sensitivity, linear range, and low detection limits for H2O2 were obtained. A sensitivity of 26.51µA/mM, linear range from (0.4 to 4.5) H2O2 mM and a LOD of 0.761 µM. The advance of this measurement system is the first step in the development process of an electrochemical biosensor for cardiovascular diseases applications.

The pollution of the planet has generated a series of problems for the environment and human health, such as the case of soils contaminated by heavy metals from batteries that are improperly disposed of in domestic garbage and sanitary landfills; Button batteries (HgO) pollute the soil, since mercury is very persistent in the environment due to its biomagnification and its great capacity to accumulate in organisms causing diseases in humans at the skin level, respiratory tract, digestive system, etc. . this being a highly toxic metal worldwide. The accumulation of Hg in soils depends on physicochemical factors such as pH, redox potential, organic matter, and clay. The main objective of this research work is to implement mercury phytoextraction in soils contaminated by button cells, using “Bamnusoideae” Bamboo, a UV-Vis spectroscopic study was carried out to determine the desorption of Hg from the contaminated soil, images of the stem were obtained, leaves and roots of bamboo without contamination and contaminated with Hg with a VE-B6 microscope, rating: 85V to 265V 50/60 Hz, Halogen Lamp: 12V 30W, and the pH of the contaminated soil was measured.

Hepsin is a type II transmembrane serine protease whose expression has been linked to greater tumorigenicity and worse prognosis in different tumors such as prostate and gastric cancer. Recently, our group described hepsin expression in the primary biopsy as a potential biomarker of thrombosis and metastasis in localized colorectal cancer patients. Here we explored the role of hepsin in this tumor. Hepsin overexpression increased Caco-2 cell migration and invasion, higher phosphorylation of Erk1/2 and STAT3 and led to more thrombin generation in plasma. Indeed, our study revealed higher plasma levels of hepsin in metastatic colorectal cancer patients, which was associated with a greater tendency toward thrombosis. By virtual screening of a FDA-approved drug library, we identified venetoclax as a potent hepsin inhibitor, reducing the metastatic and prothrombotic phenotype of Caco-2 cells, but not of other colorectal cancer cells without hepsin expression. Interestingly, pre-treating Caco-2 cells overexpressing hepsin with venetoclax reduced its in vivo invasiveness. Taken together, our results demonstrate that elevated hepsin levels correlate with a more aggressive and prothrombotic tumor phenotype. Likewise, they evidence an antitumor role for venetoclax as a hepsin inhibitor. This lays the groundwork for molecular targeted therapy for colorectal cancer.

As humanity evolves, air pollution has increased due to the various anthropogenic activities that man carries out, alienating emissions of gases and polluting particles to the environment, seriously affecting the health of living beings and the planet, since these cause Irreversible physical and chemical alterations in the environment, becoming a major problem worldwide. In Mexico there are meteorological stations that measure air quality and they are only found at fixed points, however, they do not cover all areas of the State of Mexico, the deterioration of air quality brings with it an increase in respiratory and cardiovascular diseases ; That is why this project's main objective is to build a portable electrochemical sensor using an Arduino board, using customizable software capable of quantifying and analyzing three polluting gases CO, CO2 and O3, especially in the municipality of Chimalhuacán located in the area East of the State of México.

Introduction: Three distinct phases are recognized in immune thrombocytopenia (ITP): newly diagnosed (≤3 months after diagnosis), persistent (>3–12 months after diagnosis), and chronic (>12 months)...

Extracorporeal photopheresis (ECP) is an established treatment strategy in steroid-refractory graft-versus-host disease (GVHD). This study's main objective was to analyze the clinical response and impact of ECP therapy in steroid dose reduction. A retrospective observational series of 113 patients from 7 transplantation centers was analyzed. Sixty-five patients (58%) had acute GVHD (aGVHD), and 48 (42%) had chronic GVHD (cGVHD). All ECP procedures were performed with the off-line system. The median number of procedures until achievement of initial response was 3 for both patients with aGVHD and those with cGVHD. ECP was the second-line therapy in 48% of the aGVHD cases and in 50% of the cGVHD cases. 71% of the cases of aGVHD were grade III-IV, and 69% of the cases of cGVHD were severe. The overall response rate on day 28 was 53% (complete response [CR] rate, 45%) in the patients with aGVHD and 67% (CR, 23%) in those with cGVHD. Skin was the most frequently involved organ, with a response rate of 58% (CR, 49%) in the patients with aGVHD and 69% (CR 29%) in those with cGVHD. At the end of ECP treatment, 60% of patients treated for aGVHD who responded were able to stop steroid therapy, with a median dose reduction of 100%. Significant differences in overall survival were observed for patients responding to ECP with aGVHD (hazard ratio [HR], 4.3; P.001) and with cGVHD (HR, 4.8; P = .003). Our data indicate that ECP is a valid therapeutic alternative in patients with steroid-refractory aGVHD and cGVHD, permitting significant steroid dosage reductions.

Immune thrombocytopenia is an acquired autoimmune disorder, which can affect both adults and children, characterized by lower than normal platelet counts (below 100 × 109/l). Thrombocytopenia may result in an increased risk of bleeding and puts patients at risk for serious complications. In the last decade, the multifactorial pathogenesis of ITP has become apparent, leading to greater understanding that different immune-mediated mechanisms could be involved in each patient, explaining the variable clinical presentation and response to therapies. The management of ITP patients has changed considerably in these past 10 years, but diagnosis of the disease has changed little, and remains clinical and possible only with the exclusion of other causes of thrombocytopenia. Although the search for such a test continues, to date, there is no reliable biomarker or gold-standard diagnostic test, which contributes to the high rate of misdiagnosis of the disease. This review presents the current limitations in the identification of the molecular disease underlying this disorder.

No biological predictors for the increased risk of thrombosis in patients with immune thrombocytopenia (ITP) have been identified. The aim of the study was to investigate platelet and neutrophil activation as well neutrophil extracellular trap (NET) formation in 63 ITP patients and 30 healthy volunteers. Platelet and neutrophil activation was assessed during steady state using flow cytometry analysis, and NETs were evaluated by quantitation of cell free DNA (cfDNA), and citrullinated histone-3-DNA (CitH3-DNA). Patient platelets and neutrophils showed increased CD62 and CD11b expression compared to controls (

Very few data exist on when a particular thrombopoietin-receptor agonist (TPO-RA) is favored in clinical practice for the treatment of patients with immune thrombocytopenia (ITP), about novel risk factors for vascular events (VE) with these drugs, nor about predictive factors for therapy free responses (TFR). We conducted an observational, retrospective, long-term follow-up multicenter study from November 2016 to January 2018 of 121 adult ITP patients initiating TPO-RA between January 2012 to December 2014. Data reflected that a platelet count ≤25 × 109/l at the time when the TPO-RA was initiated was associated with a 2.8 higher probability of receiving romiplostim vs. eltrombopag (P = 0.010). VE on TPO-RA was related to previous neoplasia in patients over 65 years (50% vs. 2.2%, P vs. 33%, P = 0.001). Receiving romiplostim as first TPO-RA with no subsequent TPO-RA switching was associated with a 50% likelihood of TFR after 2.9 years of therapy (3.3 years in chronic ITP patients). These real-world data help deciphering some areas of uncertainty, and offer insight into some of the most relevant challenges of ITP which may help clinicians make appropriate treatment decisions in the management of adult ITP patients with TPO-RA.

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of gene expression. Dysregulated expression of several miRNAs has been found in primary immune thrombocytopenia (ITP) suggesting that miRNAs are likely involved in the pathogenesis of ITP. We aimed to explore the differential expression of miRNAs in patients with ITP before and after starting treatment with thrombopoietin-receptor agonists (TPO-RAs) to clarify their roles in the pathophysiology of ITP, and as potential diagnostic and prognostic markers of this disorder. We performed a profiling study where 179 miRNAs were analyzed in eight ITP patients before and during treatment with TPO-RAs and in eight controls using miRNA PCR panel; 81 miRNAs were differentially expressed in ITP patients compared to controls, and 14 miRNAs showed significant changes during TPO-RA-treatment. Ten miRNAs were selected for validation that was performed in 23 patients and 22 controls using droplet digital PCR. Three miRNAs were found to be differentially expressed in ITP patients before TPO-RA-treatment compared to controls: miR-199a-5p was down-regulated (p = 0.0001), miR-33a-5p (p = 0.0002) and miR-195-5p (p = 0.035) were up-regulated. Treatment with TPO-RAs resulted in changes in six miRNAs including miR-199a-5p (p = 0.001), miR-33a-5p (p = 0.003), miR-382-5p (p = 0.004), miR-92b-3p (p = 0.005), miR-26a-5p (p = 0.008) and miR-221-3p (p = 0.023); while miR-195-5p remained unchanged and significantly higher than in controls, despite the increase in the platelet count, which may indicate its possible role in the pathophysiology of ITP. Regression analysis revealed that pre-treatment levels of miR-199a-5p and miR-221-3p could help to predict platelet response to TPO-RA-treatment. ROC curve analysis showed that the combination of miR-199a-5p and miR-33a-5p could distinguish patients with ITP from controls with AUC of 0.93. This study identifies a number of differentially expressed miRNAs in ITP patients before and after initiation of TPO-RAs with potential roles in the pathophysiology, as well as with a possible utility as diagnostic and prognostic biomarkers. These interesting findings deserve further exploration and validation in future studies.

SARS-CoV-2 infection increases the risk of thrombosis by different mechanisms not fully characterized. Although still debated, an increase in D-dimer has been proposed as a first-line hemostasis test associated with thromboembolic risk and unfavorable prognosis. We aim to systematically and comprehensively evaluate the association between thrombin generation parameters and the inflammatory and hypercoagulable state, as well as their prognostic value in COVID-19 patients. A total of 127 hospitalized patients with confirmed COVID-19, 24 hospitalized patients with SARS-CoV-2-negative pneumonia and 12 healthy subjects were included. Clinical characteristics, thrombin generation triggered by tissue factor with and without soluble thrombomodulin, and also by silica, as well as other biochemical parameters were assessed. Despite the frequent use of heparin, COVID-19 patients had similar thrombin generation to healthy controls. In COVID-19 patients, the thrombin generation lag-time positively correlated with markers of cell lysis (LDH), inflammation (CRP, IL-6) and coagulation (D-dimer), while the endogenous thrombin potential (ETP) inversely correlated with D-dimer and LDH, and positively correlated with fibrinogen levels. Patients with more prolonged lag-time and decreased ETP had higher peak ISTH-DIC scores, and had more severe disease (vascular events and death). The ROC curve and Kaplan Meier estimate indicated that the D-dimer/ETP ratio was associated with in-hospital mortality (HR 2.5; p = 0.006), and with the occurrence of major adverse events (composite end-point of vascular events and death) (HR 2.38; p = 0.004). The thrombin generation ETP and lag-time variables correlate with thromboinflammatory markers, and the D-dimer/ETP ratio can predict major adverse events in COVID-19.

In the last decade, improvements in genetic testing have revolutionized the molecular diagnosis of inherited thrombocytopenias (ITs), increasing the spectrum of knowledge of these rare, complex and heterogeneous disorders. In contrast, the therapeutic management of ITs has not evolved in the same way. Platelet transfusions have been the gold standard treatment for a long time. Thrombopoietin receptor agonists (TPO-RA) were approved for immune thrombocytopenia (ITP) ten years ago and there is evidence for the use of TPO-RA not only in other forms of ITP, but also in ITs. We have reviewed in the literature the existing evidence on the role of TPO-RAs in ITs from 2010 to February 2021. A total of 24 articles have been included, 4 clinical trials, 3 case series and 17 case reports. A total of 126 patients with ITs have received TPO-RA. The main diagnoses were Wiskott–Aldrich syndrome, MYH9-related disorder and ANKRD26-related thrombocytopenia. Most patients were enrolled in clinical trials and were treated for short periods of time with TPO-RA as bridging therapies towards surgical interventions, or other specific approaches, such as hematopoietic stem cell transplantation. Here, we have carried out an updated and comprehensive review about the efficacy and safety of TPO-RA in ITs.

Key Points In 9 unrelated families, we found 6 different TUBB1 variants, 1 of which novel, expanding the genetic spectrum of TUBB1-RT.TUBB1 variants alter β1-tubulin localization and proplatelet formation, although they show high heterogeneity in clinical presentation., Visual Abstract, β1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 β1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.

During platelet activation, arachidonic acid (AA) is released from membrane phospholipids and metabolized to thromboxane A2 (TXA2) through the actions of cyclooxygenase-1 (COX-1) and TXA2 synthase. Note, TXA2 binds to the platelet TXA2 receptor, causing shape change, secretion and platelet aggregation.1 Also, COX-1 (599aa; 70 kDa) has cyclooxygenase and peroxidase activities and it is functionally active as a homodimer, with each COX-1 monomer consisting of four highly conserved domains: an N-terminal signal peptide, a dimerization domain, a membrane-binding domain (MBD) and a large C-terminal catalytic domain2 (Figure 1A). Irreversible COX-1 inhibition by aspirin is a widely established anti-platelet therapy in cardiovascular disease., Fundación Mutua Madrileña, Grant/Award Number: AP172142019; Fundación Séneca, Grant/Award Number: 19873/GERM/15; Gerencia Regional de Salud, Grant/Award Numbers: 1647/A/17, 2061A/19; Instituto de Salud Carlos III (ISCIII) & Feder, Grant/Award Numbers: CB15/00055, PI17/01966, PI18/00598, PI20/00926, PI17/01311; Junta de Castilla y León; British Heart Foundation, Grant/Award Number: PG/17/40/33028; Ayuda a Grupos de Trabajo en Patología Hemorrágica; Premio López Borrasca 2019; Sociedad Española de Trombosis y Hemostasia.

[Background]: Ten years after their availability, thrombopoietin receptor agonists (TPO-RA) have heralded a paradigm shift in the treatment of immune thrombocytopenia (ITP). This study was aimed to analyze the implementation of current recommendations in the standard practice of adult ITP patients, and how age may influence those changes., [Methods]: We included 121 adult patients (> 65 years, n = 54; younger individuals, n = 67) who initiated treatment with TPO-RA between January 2012 and December 2014., [Results]: Patients older than 65 years treated with TPO-RA presented at diagnosis with significantly higher platelet counts, less bleeding, and a more prothrombotic profile than younger ones. The high efficacy rates of TPO-RA, preferentially used during the last decade in non-chronic phases, precluded from further therapies in the majority of ITP patients. Their administration was associated with a sharp decline in the last decade in the use of splenectomy and intravenous immunoglobulin, especially in younger ITP individuals., [Conclusion]: These results confirm (1) that there is a preferential use of TPO-RAs in elderly ITP patients with fewer bleeding complications but more unfavorable prothrombotic conditions than in younger individuals, and (2) that early use of these agents has been established as an effective therapeutic alternative to other second line therapies., This work was supported by Amgen S.A. Spain.

Major surgery is associated with an increased risk of venous thromboembolism, thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of venous thromboembolism in patients with inherited platelet disorders undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in inherited platelet disorders patients undergoing surgery at venous thromboembolism-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of inherited platelet disorders (venous thromboembolism-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to venous thromboembolism-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest venous thromboembolism-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions. Two thromboembolic events were registered, both occurring after high venous thromboembolism-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that venous thromboembolism incidence is low in patients with inherited platelet disorders undergoing surgery at venous thromboembolism-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with inherited platelet disorders undergoing invasive procedures at venous thromboembolism-risk and low-molecular-weight-heparin should be considered for major surgery. Fil: Paciullo, Francesco. Università di Perugia; Italia Fil: Bury, Loredana. Università di Perugia; Italia Fil: Noris, Patrizia. Universita Degli Studi Di Pavia; Italia Fil: Falcinelli, Emanuela. Università di Perugia; Italia Fil: Melazzini, Federica. Universita Degli Studi Di Pavia.; Italia Fil: Orsini, Sara. Università di Perugia; Italia Fil: Zaninetti, Carlo. Universita Degli Studi Di Pavia.; Italia Fil: Abdul Kadir, Rezan. Haemophilia Centre And Haemostasis Unit, Royal Free Fou; Reino Unido Fil: Obeng Tuudah, Deborah. Haemophilia Centre And Haemostasis Unit, Royal Free Fou; Reino Unido Fil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Fabris, Fabrizio. Università di Padova; Italia Fil: Rivera, Jose. Universidad de Murcia; España Fil: Lozano, Maria Luisa. Universidad de Murcia; España Fil: Butta, Nora. Hospital Universitario la Paz; España Fil: Favier, Remi. French Reference Centre For Inherited Platelet Disorder; Francia Fil: Cid, Ana Rosa. Hospital Universitario y Politecnico la Fe; España Fil: Fouassier, Marc. French Reference Centre For Inherited Platelet Disorder; Francia Fil: Podda, Gian Marco. Università degli Studi di Milano; Italia Fil: Santoro, Cristina. Università degli studi di Roma "La Sapienza"; Italia Fil: Grandone, Elvira. Casa Sollievo Della Sofferenza,s.giovanni Rotondo; Italia Fil: Henskens, Yvonne. Maastricht University Medical Centre; Países Bajos Fil: Nurden, Paquita. French Reference Centre For Inherited Platelet Disorder; Francia Fil: Zieger, Barbara. Albert Ludwigs University of Freiburg; Alemania Fil: Cuker, Adam. University of Pennsylvania; Estados Unidos Fil: Devreese, Katrien. University of Ghent; Bélgica Fil: Tosetto, Alberto. Hospital San Bortolo; Italia Fil: De Candia, Erica. Istituto Nazionale di Ricovero e Cura a Carattere Scientifico "Saverio de Bellis"; Italia Fil: Dupuis, Arnaud. French Reference Centre For Inherited Platelet Disorder; Francia Fil: Miyazaki, Koji. Kitasato University School Of Medicine; Japón Fil: Othman, Maha. Queens University; Canadá Fil: Gresele, Paolo. Università di Perugia; Italia

Severe thrombocytopenia (platelet count < 50 x 109/l) is the most frequent hematological disorder in patients with chronic liver disease, affecting 64-84 % of individuals with cirrhosis. The pathophysiological mechanisms that underlie thrombocytopenia are complex, but the reduction of thrombopoietin levels is considered to play a key role. Until recently, there was a continuous debate about the optimal management of patients with chronic liver disease and whether low platelet counts require a scheduled invasive procedure. Transfusion of platelet concentrates is considered to be the standard treatment, but has major limitations such as its short lifespan, limited efficacy and relevant adverse effects. Avatrombopag is an oral thrombopoietin receptor agonist that induces megakaryocytic proliferation and differentiation and platelet production. It has been approved by the Spanish Agency of Medicines and Health Products for the treatment of severe thrombocytopenia in adult patients with chronic liver disease before scheduled procedures, to decrease the risk of bleeding. Randomized trials have demonstrated that this agent is effective in maintaining platelet counts above 50 × 109/l over a period of more than two weeks, with a similar safety profile to the placebo. In this article, we review avatrombopag in the treatment of thrombocytopenia in patients with chronic liver disease. Furthermore, the main differences of this intervention in comparison to the previous standard of care therapy are discussed.

This study evaluated the effect of composted cow manure (CCM) on the chemical fractionation and retention degree of heavy metals (HMs) in mine tailings from Zimapan, Mexico. In a greenhouse experim...

Patients with multirefractory immune thrombocytopenia (ITP) have limited treatment options. Recent data suggest that specific anti-platelet antibodies may cause destruction of platelets by favoring platelet loss of sialic acid. In this multicenter study 35 patients with ITP, including 16 with multirefractory disease, were analyzed for antiplatelet-antibodies, thrombopoietin (TPO) levels, and platelet desialylation. In selected cases, responses to a novel treatment strategy using oseltamivir were tested. We found that antibodies against GPIbα were overrepresented in multirefractory patients compared to responders (n = 19). In contrast to conventional ITP patients, multirefractory patients exhibited a significant increased platelet activation state (granule secretion) and desialylation (RCA-1 binding) (p These findings suggest that desialylation may play a key pathogenic role in some multirefractory ITP patients, and provide diagnostic tools for the identification of such patients. Furthermore, we show that sialidase inhibitor treatment in combination with therapies that help to increase platelet production can induce sustained platelet responses in some patients with anti-GPIbα -mediated thrombocytopenia that have failed previous therapies.

Background CD56bright natural killer (NK) regulatory cells were recently shown to display a differential impact on the risk of developing extensive chronic graft-versus-host disease (GVHD). To date no study has definitively established which immune populations are most responsible for the immunomodulatory effects or response to extracorporeal photopheresis (ECP) for GVHD. Study design and methods To test the role of CD56bright NK cells in ECP, a prospective enhanced flow cytometry follow-up of immune subsets (CD19+, CD3+, CD3+/CD4+, CD3+/CD8+, CD3-/CD56+, CD3-/CD56bright , and CD3-/CD56dim ) was performed in 32 patients with GVHD who underwent 552 procedures. Results An early increase of CD56bright NK cells was found as a hallmark effect to ECP, particularly during the first 3 months of treatment. This was also supported by the ability to predict for complete responses when this increase was expressed as a higher CD56bright versus CD56dim NK cells ratio. Among the immune subsets tested, the only variable that had direct influence on response to ECP was a CD56bright/dim ratio more than 0.16 (hazard ratio [HR] 4.32, p = 0.014; HR 5.8, p = 0.007, at 2 and 3 months of ECP treatment, respectively). Conclusion These findings argue for exploring strategies for priming a CD56bright NK cell expansion during ECP and providing additional and potentially relevant data for revisiting the underpinning cellular mechanisms of ECP that could generate that expansion.

The heritable thrombocytopenias (HTs) are genetically heterogeneous rare disorders in whichreduced circulating platelet levels may be associated with nonhematological features. Amongrecently discovered HTs, DIAPH1-related disorder (D-RD; OMIM #124900) was initially reported in pedigrees with macrothrombocytopenia and hearing loss. This phenotype segregated with aheterozygous p.R1213* variant in DIAPH1, which encodes the cytoskeletal regulator diaphanoushomolog 1 (DIAPH1). This predicted truncation of the DIAPH1 C terminus diaphanous autoregulatory domain (DAD) and was proposed to confer gain-of-function, resulting in megakaryocyte (MK) cytoskeletal dysregulation and impaired proplatelet formation. Macrothrombocytopenia and hearing loss have subsequently been reported in further isolated pedigrees with DAD DIAPH1 variants,4-6 suggesting that D-RD is a distinct syndromic HT. However, other descriptions of similar DIAPH1 variants include hearing loss but not hematological findings. To provide a full phenotypic description of D-RD and the relationship with different DIAPH1 variants, we report detailed hematological findings from 5 D-RD pedigrees, including the in vitro response and clinical outcome of treatment with the thrombopoietin (TPO) receptor agonisteltrombopag.

OBJECTIVES Whole blood-derived platelet concentrates can be obtained by the platelet-rich plasma (PRP-PCs) or the buffy-coat (BC-PCs) method. Few studies have shown that BC-PCs display lower in vitro platelet activation, but scarce information exists regarding transfusion efficacy. We have performed a retrospective study assessing platelet transfusion in patients undergoing allogeneic hematopoietic cell transplantation (AHCT) in our clinic, before and after the implementation of BC-PCs. METHODS We reviewed clinical records corresponding to 70 PRP-PCs and 86 BC-PCs prophylactic transfusions, which were performed to 55 AHCT patients. Transfusion efficacy was assessed by the 24-h post-transfusion corrected count increment (24-h CCI) and bleeding events. Clinical factors affecting transfusion outcome were also investigated. RESULTS Clinical characteristics and the total number of platelet transfusions were similar among groups. Mean donor exposure was 5.8 and 5.0 in each single PRP-PCs and BC-PCs transfusion, respectively (p

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders., This study was supported by research grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundación Séneca (19873/GERM/15) and Sociedad Española de Trombosis y Hemostasia (SETH). SPW holds a British Heart Foundation chair.

Key Clinical Message Glanzmann thrombasthenia is a rare bleeding disorder that can present life‐threatening bleeding. Our patients develop antiplatelet antibodies that become refractory to any pharmacological treatment. Allogeneic hematopoietic stem‐cell transplantation is the only currently curative procedure, but has major risks mainly in adult; indeed, our patient died.

The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

Mutations in ceramide biosynthesis pathways have been implicated in a few Mendelian disorders of keratinization although ceramides are known to have key roles in several biological processes in skin and other tissues. Using whole-exome sequencing in four probands with undiagnosed skin hyperkeratosis/ichthyosis, we identified compound heterozygosity for mutations in KDSR, encoding an enzyme in the de novo synthesis pathway of ceramides. Two individuals had hyperkeratosis confined to palms and soles as well as anogenital skin, whereas the other two had more severe, generalized harlequin ichthyosis-like skin. Of note, thrombocytopenia was present in all cases. The mutations in KDSR were associated with reduced ceramide levels in skin and impaired platelet function. KDSR enzymatic activity was variably reduced in all cases resulting in defective acylceramide synthesis. Mutations in KDSR have recently been reported in inherited recessive forms of progressive symmetric erythrokeratoderma, but our study demonstrates that biallelic mutations in KDSR are implicated in an extended spectrum of disorders of keratinization in which thrombocytopenia is also part of the phenotype. Mutations in KDSR cause defective ceramide biosynthesis, underscoring the importance of ceramide and sphingosine synthesis pathways in skin and platelet biology.