A phase II study of vorinostat (MK-0683) in patients with polycythaemia vera and essential thrombocythaemia

Inhibition of histone deacetylases may be an important target in patients with myeloproliferative neoplasms. This investigator-initiated, non-randomized, open-label phase II multi-centre study included 63 patients (19 essential thrombocythaemia, 44 polycythaemia vera) from 15 centres. The primary objective was to evaluate if vorinostat was followed by a decline in clonal myeloproliferation as defined by European Leukaemia Net. Thirty patients (48%) completed the intervention period (24 weeks of therapy). An intention-to-treat response rate of 35% was identified. Pruritus was resolved [19% to 0% (P = 0·06)] and the prevalence of splenomegaly was lowered from 50% to 27% (P = 0·03). Sixty-five per cent of the patients experienced a decrease in JAK2 V617F allele burden (P = 0·006). Thirty-three patients (52% of patients) discontinued study drug before end of intervention due to adverse events (28 patients) or lack of response (5 patients). In conclusion, vorinostat showed effectiveness by normalizing elevated leucocyte and platelet counts, resolving pruritus and significantly reducing splenomegaly. However, vorinostat was associated with significant side effects resulting in a high discontinuation rate. A lower dose of vorinostat in combination with conventional and/or novel targeted therapies may be warranted in future studies.

Expansion des mégacaryocytes par HoxB4 pour accélérer la reconstitution plaquettaire

La greffe de cellules souches hématopoïétiques est parfois le seul traitement efficace contre les cancers hématologiques ainsi que plusieurs autres désordres reliés au système hématopoïétique. La greffe autologue est souvent le traitement de choix pour les patients atteints de lymphome ou de myélome. Dans ce cas, les cellules souches hématopoïétiques (CSH) du patient sont récoltées et congelées. Le patient subit ensuite des traitements de chimiothérapie et/ou radiothérapie qui éliminent les cellules malignes, mais détruisent aussi son système hématopoïétique. Ce dernier sera ensuite reconstitué par la greffe de CSH. Ces traitements ont pour conséquence de plonger le patient en état d’aplasie pour une période variant de 2 à 4 semaines. La thrombocytopénie (faible taux de plaquettes) est une complication majeure nécessitant des transfusions plaquettaires répétées et associée à une augmentation de la mortalité hémorragique post-transplantation. Il serait particulièrement intéressant de développer une thérapie accélérant la reconstitution des mégacaryocytes (MK), ce qui aurait pour effet de raccourcir la période de thrombopénie et donc de diminuer les besoins transfusionnels en plaquettes et potentiellement augmenter la survie. HOXB4 est un facteur de transcription qui a déjà démontré sa capacité à expandre les CSH et les progéniteurs multipotents (CFU-GEMM) donnant naissance aux MK. Il est donc un bon candidat pour l’expansion des progéniteurs MK. Comme la protéine HoxB4 a par contre une courte demi-vie (~1.1h), des protéines HoxB4 de deuxième génération avec une plus grande stabilité intracellulaire ont été créées (1423 (HoxB4L7A), 1426 (HoxB4Y23A) et 1427 (HoxB4Y28A)). Nous avons donc étudié la capacité d’HoxB4 sauvage et de deuxième génération à expandre les CSH, ainsi que les MK donnant naissance aux plaquettes. La surexpression rétrovirale de ces protéines HoxB4Y23A et HoxB4Y28A conduit à une expansion des progéniteurs MK murins in vitro supérieure à HoxB4-wt, 1423 et au contrôle GFP. La reconstitution plaquettaire in vivo dans un modèle murin a ensuite été évaluée par des transplantations primaires et secondaires. Les résultats révèlent que la surexpression rétrovirale des différents HoxB4 n’apporte pas de bénéfice significatif à la reconstitution plaquettaire des souris. Lorsque cultivées dans un milieu favorisant la différenciation mégacaryocytaire, le traitement de cellules CD34+ dérivées du sang de cordon ombilical avec les protéines recombinantes TATHoxB4WT ou de seconde génération n’a pas augmenté la production plaquettaire. Par contre, de manière intéressante, les cellules CD34+ provenant de sang mobilisé de patients atteints de myélome et mises en culture dans un milieu favorisant l’expansion des CSH ont montré des différences significatives dans la différenciation des progéniteurs MK en présence de la protéine recombinante TATHoxB4. La protéine HOXB4 possède donc un avenir prometteur quant à une amélioration de l’état thrombocytopénique chez les patients.

The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200000/muL) and 146 thrombocytopenic samples (less than 200000/muL). The thrombocytopenic group was further divided into 62 with platelet counts between 100000-200000/muL (Group B) and 84 samples with less than 100000 platelets/muL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis.

Characterization and quantification of blood cells from the umbilical cord of dogs

Background: Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. Objective: the aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. Methods: the blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grunwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grunwald Giemsa. Results: the MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. Conclusion: Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.

Série plaquetaŕria: Valores de referência e correlações observadas entre os parâmetros plaquetários em uma população aparentemente saudável

Mean platelet volume (MPV), platelet distribution width (PDW) and plateletcrit (PCT) platelet parameters can be important to improve the initial identification of platelet disorders. The purpose of this study is to establish reference values for the platelet count (PLT), MPV, PDW and PCT in a population of 1346 apparently healthy adults, from both sexes, as well as to evaluate the correlations among these platelet parameters and to build the necessary nomograms for the clinical interpretations of the platelet indices. The platelet parameters were analyzed in blood samples collected into dipotassium ethylenediaminetetra-acetic acid (K 2 EDTA) by an electrical aperture-impedance method. There were non-linear and an inverse correlations between MPV and PLT, and between PDW and PLT, throughout the reference range of platelet count: the change in MPV and PDW was most accentuated at the lower platelet counts. Because of this non-linear inverse correlation, MPV versus PLT and PDW versus PLT nomograms were built. These correlations among the platelet parameters on a normal population provide a better understanding of these indices and may contribute to establish the real clinical significance of these platelet parameters in many diseases.

Avaliação da reatividade peritoneal de equinos submetidos à enterotomia de cólon menor e tratados com heparina pela via subcutânea

Pós-graduação em Medicina Veterinária - FMVZ

Impacto da mistura de amaranto adicionada de arroz na proporção de 30/70% sobre a defesa antioxidante de ratos desnutridos

A desnutrição, altamente prevalente em países em desenvolvimento, é um mau antigo que aflige a humanidade. Apresenta-se como um estado de deficiência alimentar, com déficit global de proteínas e calorias, provocando menor aporte de nutrientes às células. Alguns estudos têm mostrado evidências de interação entre desnutrição e estresse oxidativo, ocasionado pelo acúmulo de espécies reativas de oxigênio que causam danos à estrutura das biomoléculas em decorrência da desregulação entre a produção de oxidante e a depleção das defesas antioxidantes. Nesse estudo foi avaliada a utilização da farinha instantânea de amaranto adicionada de arroz na proporção de 30/70% como suplemento alimentar da dieta de base do paraense usada como modelo de indução da desnutrição experimental em ratos sobre o estresse oxidativo dos animais desnutridos comparados aos controles e aos tratados com a dieta suplementada. A dieta modelo de desnutrição (DBR-PA) foi confeccionada respeitando-se as quantidades dos alimentos consumidos rotineiramente pela população do Pará, segundo inquérito alimentar realizado na década de 70 por pesquisadores da Universidade Federal do Pará, enquanto que, a dieta utilizada como tratamento foi elaborada adicionando-se a DBRPA 30% da farinha de amaranto. As análises da composição centesimal e o perfil de aminoácidos foram realizados de acordo com as normas do Instituto Adolfo Lutz (1995) e por espectrofotometria atômica. A dieta controle foi utilizada na forma que é comercializada. Para realização do estudo utilizou-se animais no pós parto imediato de mães alimentadas na gestação com dieta controle para ratos (22% de proteínas), com peso mínimo de 6 g ao nascer. No pós parto imediato as ratas mães foram divididas em 3 grupos a saber: grupo controle (22% de proteínas); grupo desnutridos (DBR-PA contendo 7,8% de proteínas) grupo 3 tratados (DBR-PA+AA) suplementada com a farinha instantânea de amaranto contendo 11,33%). No pós desmame os animais foram separados e em gaiolas individuais receberam a dieta materna específica de cada grupo até os 60 dias de vida, quando foram sacrificados e realizada a coleta de sangue para as dosagens bioquímicas (colesterol total e frações, valores hemogramas (hematimetria, leucograma e plaquetas), níveis de peroxidação lipídica e atividade da catalase. Após a coleta do sangue os animais foram submetidos à exerese do fígado para posterior análise histopatológica. Os resultados revelaram que a dieta indutora da desnutrição é um modelo de desnutrição grave comum na região norte, é hipoproteica, normocalórica, com aminoácido limitante (metionina), promoveu perda de peso nos animais desde o período de aleitamento com acentuado perda de peso nas ratas mãe e nos filhotes aos desmame (21 dias), aos 28 e 60 dias de vida (p <0,05) quando comparados aos animais tratados com amaranto e aos controles. A dieta suplementada com a farinha extrusada de amaranto promoveu ganho de peso no período do aleitamento tanto nas ratas mães (p<0,05) como nos filhotes a partir do 14º dias de uso da mesma ( p<0,05), aos 21 dias (desmame)(p<0,05) aos 28 ( p< 0,05)e 60º dias de vida (p<0,05). Os animais desnutridos consumiram mais dieta em todos os momentos avaliados quando comparados aos tratados e controles (p<0,05). Não foi observada diferença entre os grupos nos valores bioquímicos de hematimetria, leucograma, plaquetas, colesterol total e frações. Os níveis de peroxidação lipídica não apresentaram diferença estatística entre os grupos. A atividade da catalase foi maior no grupo tratado com a suplementação da farinha de amaranto quando comparado aos desnutridos.Os animais tanto os tratados com amaranto como os desnutridos apresentaram esteatose hepática e processo inflamatório dos hepatócitos.O estudo mostrou que a desnutrição imposta não ocasionou estresse oxidativo, porém a diminuição da atividade da catalase nos animais desnutridos pode ter sido ocasionado pela diminuição da síntese da catalase.

Evaluation of zebu nellore cattle blood samples using the Cell-Dyn 3500 hematology analyzer

The Cell-dyn 3500 is a multiparameter flow cytometer, which may analyze samples from several species performing several simultaneous analyses. It is able to perform white blood cells, red blood cells and platelet counts, besides differential leukocyte counts, packed cell volume and hemoglobin determination. Cell-Dyn 3500 performs total leukocyte count both optically and by impedance. The equipment may choose one or other method, based on the reliability of the results. Erythrocyte and platelet counts are determined by impedance. Leukocyte differentiation is based on an optical principle, using separation in multiangular polarized light. The objective of this study was to compare the results of complete blood count of Zebu Nellore heifers from Cell-dyn 3500, with those obtained from a semi-automated cell counter (Celm CC 510) and the manual technique. Blood samples were collected from the jugular vein in 5 mL EDTA vacuum tubes from 58 Nellore heifers, at 24 months of age. Samples were processed in parallel in the three different techniques. Results were analyzed using paired t test, Pearson's correlation and the Bland-Altmann method. There was a strong correlation for all parameters analyzed by Cell-Dyn 3500, manual method and semi-automated cell counter, except for basophils and monocytes counts. These results confirm that this analyzer is reliable for blood samples analysis of zebu cattle.

Fetal alloimmune thrombocytopenia and maternal intravenous immunoglobulin infusion

Different therapeutic approaches have been used in fetal-neonatal alloimmune thrombocytopenia, but many centers administer immunoglobulin G infusions to the pregnant woman. We studied the effect of maternal antenatal immunoglobulin infusions on fetal platelet counts in pregnancies with fetal alloimmune thrombocytopenia.

Circulating DNA and myeloperoxidase indicate disease activity in patients with thrombotic microangiopathies

Thrombotic microangiopathies (TMAs) are a group of life-threatening disorders characterized by thrombocytopenia, fragmentation of erythrocytes, and ischemic organ damage. Genetic disorders, autoimmune disease, and cancer are risk factors for TMAs, but an additional, unknown trigger is needed to bring about acute disease. Recent studies suggest that DNA and histones are released during inflammation or infection and stimulate coagulation, thrombosis, thrombocytopenia, and organ damage in mice. We show that extracellular DNA and histones as well as markers of neutrophils are present in acute TMAs. Analysis of plasma from TMA patients of different clinical categories revealed elevated levels of DNA-histone complexes and myeloperoxidase (MPO) from neutrophil granules as well as S100A8/A9, a heterocomplex abundant in neutrophil cytosol. During therapy of thrombotic thrombocytopenic purpura, a subtype of TMAs often associated with severe ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) deficiency, plasma DNA and MPO were inversely correlated with platelet counts, and their levels indicated amelioration or exacerbation of the disease. ADAMTS13 deficiency together with increased levels of plasma DNA and MPO were characteristic for acute thrombotic thrombocytopenic purpura. A minor infection often precedes acute TMA and extracellular DNA and histones released during the inflammatory response could provide the second hit, which precipitates acute TMA in patients with pre-existing risk factors.

A simple presurgical necrotizing enterocolitis-mortality scoring system

OBJECTIVE: To assess the relationship between early laboratory parameters, disease severity, type of management (surgical or conservative) and outcome in necrotizing enterocolitis (NEC). STUDY DESIGN: Retrospective collection and analysis of data from infants treated in a single tertiary care center (1980 to 2002). Data were collected on disease severity (Bell stage), birth weight (BW), gestational age (GA) and pre-intervention laboratory parameters (leukocyte and platelet counts, hemoglobin, lactate, C-reactive protein). RESULTS: Data from 128 infants were sufficient for analysis. Factors significantly associated with survival were Bell stage (P<0.05), lactate (P<0.05), BW and GA (P<0.01, P<0.001, respectively). From receiver operating characteristics curves, the highest predictive value resulted from a score with 0 to 8 points combining BW, Bell stage, lactate and platelet count (P<0.001). At a cutoff level of 4.5 sensitivity and specificity for predicting survival were 0.71 and 0.72, respectively. CONCLUSION: Some single parameters were associated with poor outcome in NEC. Optimal risk stratification was achieved by combining several parameters in a score.

International Society on Thrombosis and Haemostasis score for overt disseminated intravascular coagulation predicts organ dysfunction and fatality in sepsis patients

We evaluated the score for disseminated intravascular coagulation (DIC) recently published by the International Society for Thrombosis and Haemostasis (ISTH) in a well-defined series of sepsis patients. Thirty-two patients suffering from severe sepsis and eight patients with septic shock were evaluated following the ISTH DIC score. Fibrin monomer and D-dimer were chosen as fibrin-related markers (FRM), respectively. DIC scores for nonsurvivors (n = 13) as well as for septic shock patients were higher (P < 0.04) compared with survivors and patients with severe sepsis, respectively. Using fibrin monomer and D-dimer, 30 and 25% of patients suffered from overt DIC. Overt DIC was associated with significantly elevated thrombin-antithrombin complexes and plasminogen activator inhibitor type-1 levels as well as with significantly lower factor VII clotting activity. Patients with overt DIC had a significantly higher risk of death and of developing septic shock. Since more than 95% of the sepsis patients had elevated FRM, the DIC score was strongly dependent on prolongation of the prothrombin time and platelet counts. The ISTH DIC score is useful to identify patients with coagulation activation, predicting fatality and disease severity. It mainly depends on the prolongation of the prothrombin time and platelet counts.

Dosing lepirudin in patients with heparin-induced thrombocytopenia and normal or impaired renal function: a single-center experience with 68 patients

The recommended dose (bolus 0.4 mg/kg followed by 0.15 mg/kg per hour) of lepirudin, a direct thrombin inhibitor licensed for treatment of heparin-induced thrombocytopenia (HIT), is too high. Starting in 2001, we omitted the bolus and reduced maintenance dose by at least one-third. Analyzing 53 HIT patients treated between January 2001 and February 2007, we observed that therapeutic anticoagulation intensity already 4 hours after lepirudin start had been reached with the following initial lepirudin doses (median): 0.078 mg/kg per hour [creatinine clearance (CrCl) more than 60 mL/min], 0.040 mg/kg per hour (CrCl 30-60 mL/min), and 0.013 mg/kg per hour (CrCl < 30 mL/min). The efficacy of this treatment was documented by increasing platelets and decreasing D-dimers. Based on this experience, we derived a lepirudin dosing regimen, which was prospectively evaluated treating 15 HIT patients between March 2007 and February 2008. We show that omitting the initial lepirudin bolus and administering 0.08 mg/kg per hour in patients with CrCl more than 60 mL/min, 0.04 mg/kg per hour in patients with CrCl 30-60 mL/min, and 0.01 to 0.02 mg/kg per hour in those with CrCl less than 30 mL/min is efficacious and safe, as documented by increasing platelet counts, decreasing D-dimer levels, and rare thrombotic (1 of 46) and major bleeding (4 of 46) complications.

Does point of care prothrombin time measurement reduce the transfusion of fresh frozen plasma in patients undergoing major surgery? The POC-OP randomized-controlled trial

BACKGROUND: Bleeding is a frequent complication during surgery. The intraoperative administration of blood products, including packed red blood cells, platelets and fresh frozen plasma (FFP), is often live saving. Complications of blood transfusions contribute considerably to perioperative costs and blood product resources are limited. Consequently, strategies to optimize the decision to transfuse are needed. Bleeding during surgery is a dynamic process and may result in major blood loss and coagulopathy due to dilution and consumption. The indication for transfusion should be based on reliable coagulation studies. While hemoglobin levels and platelet counts are available within 15 minutes, standard coagulation studies require one hour. Therefore, the decision to administer FFP has to be made in the absence of any data. Point of care testing of prothrombin time ensures that one major parameter of coagulation is available in the operation theatre within minutes. It is fast, easy to perform, inexpensive and may enable physicians to rationally determine the need for FFP. METHODS/DESIGN: The objective of the POC-OP trial is to determine the effectiveness of point of care prothrombin time testing to reduce the administration of FFP. It is a patient and assessor blind, single center randomized controlled parallel group trial in 220 patients aged between 18 and 90 years undergoing major surgery (any type, except cardiac surgery and liver transplantation) with an estimated blood loss during surgery exceeding 20% of the calculated total blood volume or a requirement of FFP according to the judgment of the physicians in charge. Patients are randomized to usual care plus point of care prothrombin time testing or usual care alone without point of care testing. The primary outcome is the relative risk to receive any FFP perioperatively. The inclusion of 110 patients per group will yield more than 80% power to detect a clinically relevant relative risk of 0.60 to receive FFP of the experimental as compared with the control group. DISCUSSION: Point of care prothrombin time testing in the operation theatre may reduce the administration of FFP considerably, which in turn may decrease costs and complications usually associated with the administration of blood products. TRIAL REGISTRATION: NCT00656396.

THE ROLE OF PLATELETS IN OVARIAN CARCINOMA

Platelets represent one of the largest storage pools of angiogenic and oncogenic growth factors in the human body. The observation that thrombocytosis (platelet count >450,000/uL) occurs in patients with solid malignancies was made over 100 years ago. However, the clinical and biological implications as well as the underlying mechanism of paraneoplastic thrombocytosis associated with ovarian carcinoma remains unknown and were the focus of the current study. Following IRB approval, patient data were collected on 619 patients from 4 U.S. centers and used to test associations between platelet count at initial diagnosis, clinicopathologic factors, and outcome. In vitro effects of plasma-purified platelets on ovarian cancer cell proliferation, docetaxel-induced apoptosis, and migration were evaluated using BrdU-PI flow cytometric and two-chamber chemotaxis assays. In vivo effects of platelet depletion on tumor growth, proliferation, apoptosis, and angiogenesis were examined using an anti-platelet antibody (anti-mouse glycoprotein 1ba, Emfret) to reduce platelets by 50%. Complete blood counts and number of mature megakaryocytes in the spleen and bone marrow were compared between control mice and ovarian cancer-bearing mice. Plasma levels of key megakaryo- and thrombopoietic factors including thrombopoietin (TPO), IL-1a, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, stem cell factor, and FLT-3 ligand were assayed in a subset of 150 patients at the time of initial diagnosis with advanced stage, high grade epithelial ovarian cancer using immunobead-based cytokine profiling coupled with the Luminex® xMAP platform. Plasma cytokines significantly associated with thrombocytosis in ovarian cancer patients were subsequently evaluated in mouse models of ovarian cancer using ELISA immunoassays. The results of human and mouse plasma cytokine profiling were used to inform subsequent in vivo studies evaluating the effect of siRNA-induced silencing of select megakaryo- and thrombopoietic cytokines on paraneoplastic thrombocytosis. Thirty-one percent of patients had thrombocytosis at initial diagnosis. Compared to patients with normal platelet counts, women with thrombocytosis were significantly more likely to have advanced stage disease (p<0.001) and poor median progression-free (0.94 vs 1.35 years, p<0.001) and overall survival (2.62 vs 4.65 years, p<0.001). On multivariate analysis, thrombocytosis remained an independent predictor of decreased overall survival. Our analysis revealed that thrombocytosis significantly increases the risk of VTE in ovarian cancer patients and that thrombocytosis is an independent predictor of increased mortality in women who do develop a blood clot. Platelets increased ovarian cancer cell proliferation and migration by 4.1- and 2.8-fold (p<0.01), respectively. Platelets reduced docetaxel-induced apoptosis in ovarian cancer cells by 2-fold (p<0.001). In vivo, platelet depletion reduced tumor growth by 50%. Staining of in vivo specimens revealed decreased tumor cell proliferation (p<0.001) and increased tumor and endothelial cell apoptosis (p<0.01). Platelet depletion also significantly decreased microvessel density and pericyte coverage (p<0.001). Platelet counts increase by 31-130% in mice with invasive ovarian cancer compared to controls (p<0.01) and strongly correlate with mean megakaryocyte counts in the spleen and bone marrow (r=0.95, p<0.05). Plasma levels of TPO, IL-6, and G-CSF were significantly increased in ovarian cancer patients with thrombocytosis. Plasma levels of the same cytokines were found to be significantly elevated in orthotopic mouse models of ovarian cancer, which consistently develop paraneoplastic thromocytosis. Silencing TPO, IL-6, and G-CSF significantly abrogated paraneoplastic thrombocytosis in vivo. This study provides new understanding of the clinical and biological significance of paraneoplastic thrombocytosis in ovarian cancer and uncovers key humoral factors driving this process. Blocking the development of paraneoplastic thrombocytosis and interfering with platelet-cancer cell interactions could represent novel therapeutic strategies.