ALBERT

Description: Abstract 1542 Poster Board I-565 Introduction Sickle cell disease (SCD) is characterized by increased oxidative stress playing an important role in the pathophysiology of hemolysis, vascular occlusion and organ damage in sickle cell patients. Sickle erythrocytes are both an important source and target of reactive oxygen species (ROS). Levels of both total and reduced form of glutathione (GSH), a major intracellular anti-oxidant, have been demonstrated to be decreased in sickle erythrocytes, despite the increased de novo synthesis of GSH in these cells. The mechanism leading to this depletion of intracellular glutathione in sickle erythrocytes is not known yet. After reaction with ROS, GSH is oxidized into its oxidized form (GSSG) and can be transported actively out of the erythrocyte. We questioned whether, during episodes of increased oxidative stress, GSSG efflux in sickle erythrocytes is higher than in normal erythrocytes. Materials and methods Erythrocytes of 10 homozygous sickle cell patients and 9 race-matched healthy controls were stimulated with 2,3-dimethoxy-l,4-naphthoquinone (DMNQ), which induces intracellular ROS generation, and hydrogen peroxide (H2O2) to stimulate GSH consumption. Intra- and extracellular levels of GSH and GSSG were measured at baseline and after 210 minutes of DMNQ and H2O2 stimulation. Results While both intra- and extracellular GSSG levels (μM) at baseline were comparable in sickle and control erythrocytes (14.5(11.5–22.7) vs. 14.3(11.6–16.3) and 0.05(0.00–0.19) vs. 0.07(0.00–0.20) respectively), GSSG levels were significantly higher in sickle erythrocytes after 210 minutes DMNQ stimulation (intracellular: 74.4(52.9–93.1) vs. 45.3(40.8–66.7),P=0.005; extracellular: 23.3(18.2–37.3) vs. 13.2(11.1–14.6),P=0.001) which suggests an increased generation of GSSG intracellularly and a resulting elevated efflux to the extracellular environment. These observations were confirmed with H2O2 stimulation of erythrocytes, showing that, while comparable at baseline, the GSSG levels were higher in sickle erythrocytes after 210 minutes stimulation (intracellular: 26.1(22.8–30.1) vs. 17.5(14.2–20.1),P=0.043; extracellular: 6.9(2.3–16.6) vs.1.2(0.6–1.6),P=0.008). In contrast to the control erythrocytes, where intracellular GSH levels remained unchanged, GSH levels decreased significantly in sickle erythrocytes during DMNQ stimulation, suggesting a limited anti-oxidative reserve capacity in SCD. Conclusion GSSG efflux in sickle erythrocytes is increased and results in net loss of intracellular glutathione, rendering sickle erythrocytes more susceptible to oxidative damage. The higher rate of GSH consumption during an episode of oxidative exacerbation in sickle erythrocytes suggests a reduced anti-oxidative reserve capacity in SCD. Disclosures No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is associated with a high incidence of ischemic stroke. SCD is characterized by hemolytic anemia, resulting in reduced nitric oxide-bioavailability, and by impaired cerebrovascular hemodynamics. Cerebrovascular CO2 responsiveness is nitric oxide dependent and has been related to an increased stroke risk in microvascular diseases. We questioned whether cerebrovascular CO2 responsiveness is impaired in SCD and related to hemolytic anemia. Transcranial Doppler-determined mean cerebral blood flow velocity (Vmean), near-infrared spectroscopy-determined cerebral oxygenation, and end-tidal CO2 tension were monitored during normocapnia and hypercapnia in 23 patients and 16 control subjects. Cerebrovascular CO2 responsiveness was quantified as Δ% Vmean and Δμmol/L cerebral oxyhemoglobin, deoxyhemoglobin, and total hemoglobin per mm Hg change in end-tidal CO2 tension. Both ways of measurements revealed lower cerebrovascular CO2 responsiveness in SCD patients versus controls (Vmean, 3.7, 3.1-4.7 vs 5.9, 4.6-6.7 Δ% Vmean per mm Hg, P 〈 .001; oxyhemoglobin, 0.36, 0.14-0.82 vs 0.78, 0.61-1.22 Δμmol/L per mm Hg, P = .025; deoxyhemoglobin, 0.35, 0.14-0.67 vs 0.58, 0.41-0.86 Δμmol/L per mm Hg, P = .033; total-hemoglobin, 0.13, 0.02-0.18 vs 0.23, 0.13-0.38 Δμmol/L per mm Hg, P = .038). Cerebrovascular CO2 responsiveness was not related to markers of hemolytic anemia. In SCD patients, impaired cerebrovascular CO2 responsiveness reflects reduced cerebrovascular reserve capacity, which may play a role in pathophysiology of stroke.

Description: Erythropoietin (EPO) has an important role in both erythropoieses and angiogenesis. EPO levels are elevated in sickle cell patients due to severe anemia, but tissue hypoxia, due to for example vaso-occlusion, may also contribute to EPO elevation. In this study we sequentially determined EPO plasma levels in HbSS sickle cell patients (n=14, 39 samples) presenting with vaso-occlusive complications at our emergency ward and the period thereafter. Patients were also requested to fill out a pain-intensity score during these events. Our results show a striking correlation between the course of vaso-occlusive events and plasma EPO levels. Vaso-occlusive crises were accompanied by impressive increments of plasma EPO levels with quick reduction of plasma EPO levels upon crisis amelioration. Changes in plasma EPO levels also closely reflected changes in pain intensity. The results of this study warrant further analysis of the potential role of EPO in the pathophysiology of sickle cell disease related complications, as well as the applicability of employing EPO levels as a marker of vaso-occlusive disease activity.

Description: Abstract 821 Introduction: Sickle cell disease (SCD) is characterized by recurrent acute vaso-occlusive painful crisis frequently leading to SCD related complications such as acute chest syndrome, stroke, multi-organ failure and even sudden death. The complex pathophysiology of the vaso-occlusive painful crisis is mediated by activation of endothelial cells, adhesion of sickled erythrocytes and neutrophils, oxidative stress, coagulation activation and increased release of inflammatory mediators, resulting in ischemic organ damage. Recently, neutrophils have been demonstrated to form neutrophil extracellular traps (NETs) upon activation. Nucleosomes and histones exposed together with neutrophil proteases, such as elastase on these NETs have been shown to kill efficiently bacteria. NET formation has been shown to propagate coagulation in sepsis and in deep venous thrombosis. In addition, nucleosomes and histones exposed on NETs have been shown to be strongly cytotoxic to endothelial cells. Beside the exposure on NETs, nucleosomes can be actively released into the circulation from dead cells. Circulating nucleosomes detected in sepsis have been reported to correlate with severity of inflammation, organ dysfunction and mortality. However, no studies are available yet on the dynamics of nucleosomes and NETs in sickle cell patients suffering from painful crisis. The aim of this case-control study was to assess plasma levels of circulating nucleosomes and human neutrophil elastase–α1-antitrypsin (EA) complexes as measure of systemic neutrophil activation, in sickle cell patients during steady state and painful crisis. Methods: Plasma levels of nucleosomes and EA as a measure of neutrophil activation were measured in 74 patients in asymptomatic state (49 HbSS/HbSβ0-thalassemia, and 25 HbSC/HbSβ+-thalassemia), 70 painful crises (53 HbSS/HbSβ°-thalassemia and 17 HbSC/HbSβ+-thalassemia) in 49 patients and in 24 HbAA healthy controls using Enzyme-Linked Immunosorbent Assay (ELISA). Results: Plasma levels of nucleosomes in both HbSS/HbSβ°-thalassemia and HbSC/HbSβ+-thalassemia patients were significantly higher during painful crisis (median; interquartile range, 20.2; 8.9 – 129.0 U/ml, P 〈 0.0001 and 11.7; 5.1 – 67.7 U/ml, P = 0.045 respectively) as compared to patients in steady state (6.0; 3.0 – 9.8 U/ml and 7.1; 4.6 – 9.6 U/ml respectively). Nucleosomes levels in healthy controls were just above the detection limit of the assay (5.0; 5.0 – 6.5) U/ml). Plasma levels of EA in HbSS/HbSβ°-thalassemia patients were significantly increased during painful crisis as compared to steady state (75.1; 56.5 – 102.4 vs. 45.7; 34.7 – 59.7 ng/ml, P 〈 0.0001). Also in HbSC/HbSβ+-thalassemia patients, EA levels were higher during painful crisis than in steady state, though the difference did not reach statistical significance (62.0; 48.0 – 96.7 vs. 50.2; 33.3 – 67.7, P = 0.051). Plasma levels of EA in healthy controls (39.9; 31.5 – 62.2 ng/ml) were comparable with those in steady state patients. In a paired analysis of 36 patients, included both during steady state and painful crisis, significant increments were observed during painful crisis in levels of both nucleosomes (from 5.0; 3.0 – 10.8 to 20.2; 6.8 – 94.3 U/ml, P 〈 0.0001) and EA (from 47.9; 36.0 – 67.6 to 70.6; 55.9 – 101.4 ng/ml, P 〈 0.0001), as compared to steady state. During painful crisis, EA levels were strongly correlated with levels of nucleosomes in both HbSS/HbSβ°-thalassemia (Spearman's rank (Sr)=0.55, P

Description: Abstract 1641 Elevated tricuspid regurgitant jet flow velocity (TRV) occurs in approximately 30% of adults with sickle cell disease (SCD) and is reported to be an independent risk factor for early death with a mortality rate as high as 40% after 4 years. We previously presented the results on a cohort of 85 consecutive ambulatory sickle cell patients that were prospectively screened for elevated TRV (2.5 m/sec) using trans-thoracic echocardiography. We aimed to determine the mortality rate in relation to TRV and associated factors in a well characterized cohort of sickle cell disease patients in the Netherlands. Follow up consisted of regular outpatient visits including laboratory testing and repeated echocardiography every 2 years. Baseline hemoglobin (Hb), lactate dehydrogenase (LDH), brain natriuretic peptide (BNP), N-terminal pro brain natriuretic peptide (NT-proBNP), asymmetric dimethylarginine (ADMA) and arginine plasma levels were related to outcome, as were baseline pulmonary function tests and coagulation studies. The prevalence of elevated TRV in the cohort at baseline was 30% (41% in HbSS/HbS0-thalassemia patients and 13% in HbSC/HbS+-thalassemia patients). Median (IQR) follow-up for the whole group was 53 months (50-57). No patients were lost to follow-up. Four patients (3 HbSS and 1 HbSβ0-thalassemia) died during follow-up. Two of these patients had an elevated TRV at baseline while the other two initially had immeasurable TRV, resulting in a death rate for patients with an elevated TRV of 8% and a ratio for early death in of 2.3 (95 percent confidence interval, 0.3 to 16.6; p=0.40) However, the two patients with immeasurable TRV at baseline had an elevated TRV (2.55 m/sec and 2.92 m/sec) on repeated echocardiography two years later. Median age of the patients who died was 48 (34-58) years compared to 29 (21-44) years of those who survived (p=0.18). Baseline plasma levels (medians (IQR)) of Hb and LDH in the deceased were 4.7 (4.3-5.0) mmol/L and 676 (575-801) U/L compared to 5.4 (4.8-6.0) and 410 (341-629) in the survivors (p=0.075 and p=0.049) respectively. Baseline median NT-proBNP plasma levels in the deceased were 278 (131-686) pg/mL and 520 (154-3957) pg/mL compared to 45 (26-94) and 91 (52-150) in the survivors (p=0.007 and p=0.03) respectively. Pulmonary function testing, coagulation studies and plasma levels of ADMA and arginine were not associated with mortality in this cohort. Conceding the relative young age of our cohort, we conclude that the mortality in sickle cell patients with elevated TRV values was far lower as compared to the published literature. Although an elevated TRV could still be an important risk factor for early death, mortality may in fact be much lower than previously reported which is in line with more recent observations by other groups. With respect to biomarkers, we confirmed that NT-pro BNP and BNP levels were significantly higher in patients who died, with a trend toward a higher rate of hemolysis as well. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2572 Poster Board II-549 Introduction: Sickle cell disease (SCD) is commonly manifested through skeletal involvement. Besides the characteristic acute musculoskeletal pain, SCD is also associated with chronic skeletal complications such as osteopenia and osteoporosis. During bone resorption, the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are released into circulation with subsequent urinary excretion. Measurements of urinary PYD and DPD could serve as valuable tools in detecting osteoporosis in the follow-up of SCD patients but perhaps also in determining the severity of bone infarction during painful crises. Therefore we compared urinary concentrations of PYD and DPD of SCD patients during asymptomatic state and painful crisis with those of race- and age-matched healthy controls. Methods: Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in SCD patients both during asymptomatic state (n=38) and painful crisis (n=27) and healthy controls with normal HbA hemoglobin (n=25) using high performance liquid chromatography (HPLC). Results: PYD and DPD concentrations were higher in asymptomatic SCD patients compared to controls ((54.8 (41.5–68.6) vs. 44.1 (37.7–49.9),P=0.005 and 11.6 (9.3–15.2) vs. 8.5 (6.8–10.4),P=0.004 respectively), with further increments during painful crisis (63.3 (51.8–76.0),P=0.041 and 15.3(13.0–21.5),P=0.003 respectively). In the asymptomatic patients levels of PYD and DPD were significantly correlated to the degree of hemolysis. Conclusion: In sickle cell patients bone resorption is increased and significantly correlated to the degree of hemolysis, compatible with their susceptibility to osteopenia and osteoporosis. Measurement of pyridinoline and deoxypyridinoline could have additional value as biomarkers of osteoporosis in SCD. During painful crises a further increment in bone degradation was observed. Disclosures: No relevant conflicts of interest to declare.

Description: A hypercoagulable state exists in hyperthyroidism, but the association with venous thrombosis (VT) is not fully explored. We aimed to investigate VT risk for different plasma levels of thyroid hormones and thyroid antibodies. We used a case-control study on leg vein thrombosis conducted between September 1999 and August 2006 at the Academic Medical Center, Amsterdam, The Netherlands. Parameters of thyroid function were assessed in 190 cases (mean age, 57 years; range, 19-90 years) and 379 sex-matched controls (mean age, 56 years; range, 18-93 years). Odds ratios (ORs) and 95% confidence intervals (CIs) for VT risk were estimated according to several cutoff levels derived from plasma levels observed in controls. We found the risk of VT to gradually rise with increasing levels of free thyroxine (FT4). In the absence of traditional acquired risk factors, FT4 levels above 17 pmol/L yielded a sex- and age-adjusted OR of 2.2 (95% CI, 1.2-4.2) for deep VT, which further increased up to an OR of 13.0 (95% CI, 1.1-154.1) for FT4 levels above reference range. Our data suggest increasing levels of FT4 to be a risk factor for VT and may have implications for both the prevention and management of this disease.