Search

Introduction: Circulating microparticles (MP) have been described in sickle cell anaemia (SCA); however, their interaction with endothelial markers remains unclear. We investigated the relationship between MP, protein C (PC), free protein S (PS), nitric oxide (NO), endothelin-1 (ET-1) and adrenomedullin (ADM) in a large cohort of paediatric patients.Method: A total of 111 children of African ethnicity with SCA: 51 in steady state; 15 in crises; 30 on hydroxyurea (HU) therapy; 15 on transfusion; 17 controls (HbAA) of similar age/ethnicity. MP were analysed by flow cytometry using: Annexin V (AV), CD61, CD42a, CD62P, CD235a, CD14, CD142 (tissue factor), CD201 (endothelial PC receptor), CD62E, CD36 (TSP-1), CD47 (TSP-1 receptor), CD31 (PECAM), CD144 (VE-cadherin). Protein C, free PS, NO, pro-ADM and C-terminal ET-1 were also measured.Results: Total MP AV was lower in crisis (1.26×106 ml−1; 0.56–2.44×106) and steady state (1.35×106 ml−1; 0.71–3.0×106) compared to transfusion (4.33×106 ml−1; 1.6–9.2×106, p0.9, p

Sickle cell anaemia (SCA) is the consequence of abnormal haemoglobin production due to an inherited point mutation in the β-globin gene. The resulting haemoglobin tetramer is poorly soluble when deoxygenated, and when this is prolonged, intracellular gelation of sickle haemoglobin occurs, followed by haemoglobin polymerisation. If many cycles of sickling and unsickling occur, the red cell membrane will be disrupted leading to haemolysis and vaso-occlusive events. Recent studies have also shown that leucocyte adhesion molecules and nitric oxide (NO) depletion are involved in endothelial damage. New insights in SCA pathophysiology and vascular biology have shown that cell-derived microparticle (MP) generation is also involved in the vaso-occlusion. Endothelial damage is perpetuated by impaired production or increased consumption of protective modulators such as protein C, protein S and NO. New therapeutic interventions should address these aspects of SCA pathogenesis. To date, the only US-FDA-approved therapy to prevent painful vaso-occulsive episodes is hydroxyurea that reduces haemoglobin polymerisation in sickle cells by increasing the production of foetal haemoglobin and L-glutamine. However, several new drugs have been tested in the last years in randomised clinical trials. We here report an update on the current status of knowledge on SCA.

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU + ASA, 15 on ANA + ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA + ASA (P 0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P 0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

Sickle cell disease (SCD) and sickle cell trait (SCT) are found most frequently in individuals of African, Middle Eastern and Indian ethnicity. Population migration has made this disease more common worldwide, including Ireland. We present an overview of this disease, focusing on management and practical implications for dental practitioners.

Abstract 5193 Essenthial thrombocythemia (ET) patients are at risk of developing an arterious or venous thrombotic event. Recent studies have shown that there is no clear association between platelet (PLT) count and thrombotic events, but that white cell count plays a role. Qualitative PLT abnormalities, activation of endothelial cells (EC) and PLT may be involved in these processes. Following activation of blood cells, plasma membrane vescicles are released, these are known as microparticles (MP) ( Total-MP were increased in all patients groups compared to controls (p This study confirms previous findings that MP are present in ET. We also showed that MP are affected by treatment and that MP are mainly PMP and express CD62P and CD36, a marker of immature PLT, which suggests that reticulated PLT partecipate in these processes. We also showed that HU increase NO and ADM levels, while AN reduces ET-1. As NO and ET-1 have antagonistic actions on endothelial cells, we suggest that HU and AN may have overlapping effects on endothelium, although using different biochemichal pathways. The finding that treatment influences PC1 (PMP and EMP) supports the idea that in ET, treatment impacts directly on MP. The finding that NO and RMP have a negative loading within PC4, suggests that they have an antagonist-action to PMP and EMP in ET. Disclosures: Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria.

Abstract 3157 Poster Board III-94 Introduction Transfusion circulatory overload (TACO) is a complication of transfusion, often confused with transfusion acute lung injury (TRALI), and is probably under-reported. The pathophysiology is believed to be caused by increased hydrostatic blood pressure leading to fluid leakage in the alveolar space, emulating congestive cardiac failure. We report on a large cohort of TACO cases in Ireland. Methods All consecutive TACO cases reported to the National Haemovigilance Office of Ireland between 2000-2008 were retrospectively reviewed. The incidence of TACO was calculated on units issued from the Irish Blood Transfusion Service (IBTS). Comparative analysis of patient demographic and clinical data was made using Fisher's exact test. Results 179 TACO cases were reviewed. 149 (83%) involved red cell concentrate (RCC), platelets (PLT) n=4 (2%); fresh frozen plasma (FFP) /solvent detergent plasma (SDP) n=16 (8%); multiple components n=10 (6%). During the study period, a total 1,614,973 blood components (RCC, FFP, SDP and PLT) were issued corresponding to a TACO incidence of 1/10.000 of all components issued. TACO incidence per RCC, platelets and plasma issued was 1/8000, 1/48000 and 1/15000 respectively, excluding the cases where multiple components were transfused. While the majority of patients developing TACO were elderly (> 70years)(n= 120, 67%), up to eight cases (4%) were reported in young patients ( 70 years, all of whom had underlying cardio–vascular conditions. Four (22%) patients died, one death which was possibly attributed to transfusion, and a further three patients (17%) required admission to intensive care. Four (22%) cases involved young female patients (age < 30 years) who had no underlying cardiovascular condition, but one patient had asthma. These patients were being treated for obstetric bleeding, and all recovered. Conclusion This study reports a TACO incidence of 1 /10 000 units issued from the IBTS. Although this incidence is based on units issued rather than transfused, it probably reflects significant underreporting, and perhaps even under-recognition. This study identified TACO has significant impact on patient outcome with approximately 8% (15) of patients suffering significant morbidity and a further 2% (5) of patients dying following onset of TACO. Finally, patients receiving large volume transfusion in emergency situations even young patients are at risk of developing TACO. Disclosures No relevant conflicts of interest to declare.

Nitric Oxide (NO) and endothelin-1 (ET-1) are key modulators of endothelial vessels and actively participate in the pathophysiology of vaso-occlusive events. Sickle cell anemia (SCA) is an important model for the study of blood cells/endothelium interactions. In SCA vaso-occlusive events may be severe and cumulative. Adrenomedullin (ADM) is a strong hypotension-inducer and mediates diuresis, natriuresis and vasodilation. Adrenomedullin levels are raised in chronic cardiovascular conditions. The aim of this study was to investigate the role of adrenomedullin in SCA and its relationship to endothelin-1 and nitric oxide. A cohort of 111 children with HbSS SCA was studied with 18 age-matched controls of sub-Sahara African ethnicity. The patients were in four clinical groups: steady state (n=55, mean age 5.3 years), patients undergoing chest crisis (n=9, mean age 5.3 years), on hydroxyurea (n=30, mean age 7.2 years), on transfusion therapy (n=17, mean age 6.5 years). A third cohort of patients with HbSC genotype (n=13, mean age 6 years) all in steady state was also included. Pro-adrenomedullin and c-terminal endothelin-1 precursor (CT-proET-1) were measured by coated tube assays according to the method by Morgenthaler et al (Clin Chem.2005;51:1823–29) and Papassotiriou et al (Clin Chem.2006;52(6):1144–51), nitric oxide was quantified according to the Griess reaction. Results (median and interquartile range) are shown in table 1. Overall pro-adrenomedullin levels were raised in chest crisis: 0.38 nmol/l (0.31–0.49) vs steady state: 0.27 nmol/l (0.25–0.32; p Table 1. ADM, ET-1, NO and hematological/biochemical values in SCA patients and controls. IR= interquartile range. Figure 1. ADM and NO levels in sickle cell patients and controls. Bars indicate median and interquartile range. Figure 1. ADM and NO levels in sickle cell patients and controls. Bars indicate median and interquartile range.

Microparticles (MP) originate from blebbing and shedding from cell membrane surfaces in physiological and pathological conditions. Increased levels are generated by a number of mechanisms including platelet activation, vascular endothelial damage, thrombin activity, C5b-9 activation, and PF4-heparin-antibody interaction. Increased circulating MP have been described in patients with sickle cell anemia (SCA). Elevated monocyte-derived MP expressing tissue factor have been reported in patients in crisis. The lack of a standardised method for MP quantification remains problematic. We measured MP (numbers and functional markers), protein C and free protein S, in a large cohort of pediatric patients to investigate the role of MP in SCA and their relation to protein C and free protein S plasma levels. One hundred and eleven children of sub-Saharan African ethnicity with SCA (hemoglobin (Hb) SS) were studied: 51 without previous history of crisis in steady state (mean age 5.3 years); 15 in crisis (9 chest crises, 6 other, mean age 5.4 years); 30 on hydroxyurea (mean age 7.2 years); 15 on transfusion therapy (mean age 6.5 years); 17 children of sub-Saharan African ethnicity of similar age (mean age 4.6 years) were used as control group (Hb AA). MP were analyzed by flow cytometry, according to Biró et al (J Thromb Haemost.2004; 2(10):1842–51), using Annexin V and antibodies against, CD61, CD42a, CD62P (P-selectin), CD235a, CD14, CD142 (tissue factor), CD201 (endothelial protein C receptor or EPCR), CD62E (E-selectin), CD36 (thrombospondin or TSP-1), CD47 (TSP-1 receptor), CD31 PECAM (platelet-endothelial cellular adhesion marker), CD144 (VE-cadherin). Protein C (chromogenic) and free protein S (latex based assay) were measured in all subjects. Correlation was measured by Pearson Rank test, and comparisons between groups were analyzed by Mann-Whitney test. Total MP AV were lower in crisis (1.26 × 106/ml; 0.56–2.44 × 106) and steady state (1.35 × 106/ml; 0.71– 3.0 × 106) compared to transfusion (4.33 × 106/ml; 1.6–9.2 × 106p0.9, p