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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.

Traditional laboratory markers of disease severity (Hb concentrations, HbF%, LDH concentrations and leukocyte counts) are not powerful enough to identify individuals with sickle cell disease (SCD) at high risk of complications. In several disease states, the number of circulating endothelial cells (CEC) is indicative of the extent of vascular disease and several small studies employing cumbersome techniques have shown increased CEC to occur in SCD as well. In this pilot study we determined if, by employing the CellSearch system, elevated CEC can be detected in SCD and whether the CEC count is related to the presence of organ damage, painful crisis frequency, traditional laboratory markers of disease severity as well as markers of endothelial activation. Peripheral blood was collected from 15 sickle cell patients and 15 healthy sex, age and race matched HbAA controls. Cells positive for CD146 (present on endothelial cells) are immuno-magnetically isolated and stained with nuclear DAPI and anti-CD105 (present on endothelial cells) and anti-CD45 (pan-leukocyte marker). Within the CD146 enriched cell suspension, cells meeting the DAPI+/CD146+/CD105+/CD45− criteria after image cytometry are designated CEC. CEC counts were significantly higher in sickle cell patients (12 cells/mL, IQR 8–29) as compared to controls (4 cells/mL, 3–10) (p=0.007). CEC counts were significantly higher in patients with pulmonary hypertension (PHT) (p=0.02), and increased with the presence of number of affected organs as well (0–4 involved organs, p=0.03), with the number of affected organs being the most important determinant of increased CEC numbers in SCD (R2=0.72, P=0.001). No statistically significant correlation was detected between CEC counts and markers of endothelial activation (serum sVCAM-1 and plasma VWF:Ag levels). CEC counts did not differ between patients on hydroxyurea and those who did not use hydroxyurea. No statistically significant correlations were detected between CEC counts and painful crisis frequency, Hb, HbF%, leukocyte counts, LDH concentrations. In conclusion, elevated CEC’s can be demonstrated in the clinically asymptomatic state in patients with sickle cell anemia with a validated and automated technique and, for the first time, a relation of CEC counts to SCD related organ damage is demonstrated. Furthermore, the extent of EC activation (as assessed by sVCAM-1 and VWF:Ag levels) is not necessarily related to the extent of EC damage (as assessed by CEC counts).

Inflammation and angiogenesis are of importance in the pathophysiology of sickle cell disease (SCD). Recently, the chemokine Stromal Derived Factor-1 (SDF-1) has been shown to be a key mediator of angiogenesis and inflammation. In this study we determined serum SDF-1 levels in consecutive adult sickle cell patients during the clinically asymptomatic state as well as during painful crisis. Serum SDF-1 levels were significantly elevated in HbSS/HbSβ0-thalassemia patients ([n=39], 3805 pg/mL (2121–6790)) as opposed to HbSC/HbSβ+-thalassemia patients ([n=18], 2405 pg/mL (1365–3047)) and healthy HbAA controls (n=25, 2623 pg/mL (2435–2988)) (p=0.04). During painful crisis SDF-1 levels increased significantly in both HbSS/HbSβ0-thalassemia ([n=24]) 26040 pg/mL (20135–27960)) and HbSC/HbSβ+-thalassemia patients ([n=5], 12666 pg/mL (3936–24453)) (p

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles plays an important role in the etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells, exhibits high affinity for PS and has been shown to inhibit microparticle-induced PS-mediated thrombin generation. We determined plasma annexin A5 levels and microparticles in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty five HbAA blood donors served as controls. Both annexin A5 levels and microparticle numbers were highest in HbSS patients (5.7 ng/mL IQR 3.7–7.6 and 37.9 nM, IQR 31.9–69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7–7.6 and 20.9 nM IQR10.9–29.6) and healthy controls (2.5 ng/mL, IQR 1.4–4.4 and 13.1 nM, IQR 9.5–18.5) (p=0.01 and p

Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenously produced inhibitor of nitric oxide (NO) synthase, are elevated and related to endothelial activation in clinically asymptomatic patients with sickle cell disease (SCD). However, ADMA concentrations have not been defined during acute vaso-occlusive complications. We determined plasma ADMA and arginine concentrations in 96 samples (40 collected in the clinically asymptomatic state and 56 collected at presentation with a painful crisis) of 44 HbSS patients (mean age 32±13 years, m:f=23:21) and in 39 samples (22 collected in the clinically asymptomatic state and 17 collected at presentation with a painful crisis) of 22 HbSC patients (mean age 30±14 years, m:f=10:12). Thirty-five race, sex and age matched HbAA blood donors served as healthy controls. Plasma ADMA concentrations were elevated in asymptomatic sickle cell patients as compared to healthy controls (HbSS: mean 0.7±0.1 umol/L, HbSC mean 0.5±0.1 umol/L, HbAA mean 0.4±0.1 umol/L; p

Sickle cell disease (SCD) is characterised by acute complications such as the painful vaso-occlusive crisis, acute chest syndrome and stroke. Although these acute complications are well known and mostly easy recognised, the impact of sickle cell-related chronic end-organ damage on outcome may well be underappreciated. In fact, often SCD severity is assessed. by the frequency of acute vaso-occlusive crises. However, accumulating chronic organ damage (such as renal failure and pulmonary hypertension) is nowadays recognized as an important determinant of disease morbidity as well. The aim of this study is to evaluate the prevalence of sickle cell-related complications and organ damage and their relation to SCD genotype and to established laboratory parameters of SCD morbidity in a tertiary teaching hospital. Furthermore, relations between complications and different forms of organ damage will also be determined. Sickle cell patients were consecutively and systematically screened for manifestations of chronic organ damage and their history of complications related to SCD and/or SCD-related treatment was retrospectively studied from their medical records. Organ damage and the history of sickle cell-related complications were expressed as a cumulative disease morbidity score (DMS). Every form of organ damage or complication scores 1 point and is divided by the number of screened items resulting in a maximal DMS of 1. Correlations were analysed with the Chi-Square test. All adult sickle cell patients (HbSS, HbSβ and HbSC) were included in our study (n=99). Six patients were excluded from analysis because of incomplete data (less than 7 of the 12 items). Prevalence of specific organ damage and complications Total HbSS HbSβ HbSC P value N 93 49 16 28 Age (year) 28 (22–39) 27 (21–41) 35 (25–41) 28 (20–34) 0.324** Female (%) 65 62 56 71 0.567 Disease Morbidity Score 0.11 (0–0.25) 0.18 (0.09–0.33) 0.09 (0–0.19 0.10 (0–0.17 0.010** Organ damage/ complications (%) Microalbuminuria 22 33 0 15 0.063 Renal failure 10 15 0 7 0.201 Pulmonary hypertension 25 40 0 7 0.006 Retinopathy 41 28 30 65 0.016 Perceptive hearing loss 12 9 27 6 0.205 Iron overload 10 17 0 4 0.063 Cholelithiasis 51 66 43 27 0.009 Avascular osteonecrosis 12 17 13 4 0.242 Leg ulcers 5 11 0 0 0.089 Acute chest syndrome 24 31 25 11 0.128 Frequent vaso-occlusive crises (>2/yr) 14 17 19 4 0.374 Stroke 6 13 0 0 0.053 Numbers are percentages or medians (iqr). * Two-sided Chi-square tests **Two sided Kruskal-Wallis test The DMS correlated significantly with c-reactive protein (R= 0.27, P=0.013), blood urea nitrogen (R=0.422, P>0.001) and hemoglobin (R= −0.326, P=0.003). No statistically significant correlation with leukocyte counts or HbF% was demonstrated. These data underscore the importance of systematic evaluation of all patients with SCD in the outpatient setting since many of the sickle cell-related complications may otherwise go unnoticed, thereby delaying the institution of potential therapeutic measures. Importantly, the lack of any significant association between the frequency of vaso-occlusive crises and manifest sickle cell-related organ damage illustrates its limited value as a marker of disease severity.

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.

Endothelial cell activation occurs continuously in sickle cell disease (SCD). As VWF and especially the ultra large VWF multimers, released from activated endothelial cells, can serve as bridging proteins between the vessel wall and sickle red blood cells, we determined ADAMTS13 (the vWF-cleaving protease) activity as well as VWF:Rcof and VWF:Ag levels in SCD. Fourteen sickle cell patients (13 HbSS, 1 HbSC; 39 samples collected) and 9 HbAA controls were included. VWF:Rcof and VWF:Ag were assessed by commercially available assays, ADAMTS13 activity was determined by quantitative immunoblotting of proteolysed VWF-substrate. VWF:Rcof and VWF:Ag levels were higher in patients, as compared to controls. ADAMTS13 activity ranged from 45–128% in asymptomatic patients, from 38–205 in patients during acute vaso-occlusive events (VOE) and from 43-75% in controls. The ratios of ADAMTS13 to vWF:Ag levels and activity were lower in patients (SCD asymptomatic: 0.44 [interquartile range 0.34–0.55], SCD acute VOE: 0.29 [0.21–0.43], HbAA: median 0.61 [0.33–1.23], Kruskar Wallis test p=0.003). The ADAMTS13 - VWF:Rcof and the ADATS13 - VWF:Ag ratios dropped during acute vaso-occlusive events in 3 of 6 patients. In patients from whom multiple samples were collected (n=4), ADAMTS13 activity and VWF:Ag levels (and to a lesser extent VWF:Rcof) were inversely related. One patient showed no change in ADAMTS13 activity despite strong increments in VWF:Ag and VWF:Rcof during vaso-occlusive pain crises prior to his death (pulmonary embolism). VWF:Ag levels at initial presentation of acute VOE were related to the duration of pain (rs=0.53, p=0.02), whereas the ratios of ADAMTS13 to VWF:Ag levels and ADAMTS13 to VWF:Rcof were inversely related hereto (rs=−0.67, p=0.002, rs=−0.53, p=0.02 respectively). These data demonstrate that ADAMTS13 activity is related to VWF:Ag levels and VWF:Rcof in SCD and that the role of ADAMTS13 and VWF in the pathophysiology of SCD deserves further evaluation.