Disease Severity In Sickle Cell Anemia Research Articles

Adiponectin and Disease Severity in Sickle Cell Anemia Patients Attending a Tertiary Health Institution in Nnewi, Southeast Nigeria.

Background: Hemoglobin polymerization in sickle cell anemia (SCA) leads to abnormally rigid and adhesive erythrocytes that obstruct blood vessels, leading to poor tissue perfusion, hence provoking inflammation and damage of surrounding tissues. Adiponectin, a protein hormone, presumptively has anti-inflammatory characteristics, hence may be an important therapeutic target in SCA. Aim: The aim of the study was to evaluate the status of adiponectin and its correlation with disease severity in SCA. Patients and Methods: A total of 84 subjects were recruited for the study comprising 34 homozygous sickle cell (HbSS) subjects (25 in the steady state and nine in the resolving crisis state) and 50 controls (25 heterozygous sickle cell [HbAS] and 25 hemoglobin phenotype AA subjects). The hemoglobin phenotype, adiponectin levels, and full blood counts were evaluated. Anthropometric measurements were also conducted. Results: A significant difference was observed in the mean body mass index between the different hemoglobin phenotype groups and also between the SCA in crisis resolution patients and the control group (p < 0.05). There was no significant difference in the median serum levels of adiponectin in the different hemoglobin phenotype groups and between SCA patients in the steady state compared with those in the crisis resolution state. Also, there was no correlation between disease severity and adiponectin in SCA patients in the steady state (p = 0.87). Conclusion: Our study seems to suggest that in our data set of sickle cell anemia patients in the steady state, adiponectin does not constitute part of the endocrinopathy that affects these patients.

Splenomegaly in Adolescents and Adults with Sickle Cell Anemia in Tanzania Is Associated with Less Severe Disease

▪Background: Identifying markers of disease severity in patients with sickle cell anemia (SCA) could help to improve patient management and inform discovery of new therapies. Tanzania has one of the largest single-centre for clinical and research programs in the world. We observed that some individuals with SCA have splenomegaly that persists into adolescence and beyond. Palpable spleen in 10% of individuals attending the outpatient clinics at Muhimbili National Hospital has previously been described (Makani et al, 2010). Splenomegaly was palpable in more than one-third of 10-year olds in an observational study of children with SCA in Kilifi (Sadarangani et al, 2009). These individuals do not fit the natural history of the disease pathogenesis as most patients with SCA may have suffered splenic infarction leading to auto-splenectomy by the time they are adults. This study sought to better understand the clinical implications of persistent splenomegaly by investigating its prevalence and clinical correlates.Methods: This was a descriptive cross-sectional study involving individuals with SCA aged ten years and older at steady-state who attended clinics in the Muhimbili Sickle Cell (MSC) program at Muhimbili National Hospital, Tanzania, between December 2015 and February 2016. Patients with SCA were screened for splenomegaly by physical exam. Abdominal ultrasound was done in all recruited individuals to document findings on the spleen, liver and biliary tree respectively. Splenomegaly was defined as any palpable spleen below the left coastal margin. Socio-demographic and clinical information were obtained using a structured questionnaire. Laboratory evaluation included automated complete blood count, reticulocyte count, peripheral smear for identification and quantification of Howell-Jolly bodies, rapid diagnostic test to detect malaria, and markers of hemolysis (lactate dehydrogenase and bilirubin). Records from the Muhimbili Sickle Cohort database were used to confirm the SCD status and obtain fetal hemoglobin levels. Chi-square test was used for analysis of categorical variables and independent student t -tests were used to compare sample means for continuous variables.Results:A total of 194 individuals between the ages of 10 - 42 years (median 17 years) were recruited; 60.3% were females. Splenomegaly by palpation was observed in 9 individuals (5%) with a slight male predominance. Meanwhile, a spleen was present in 86 (44.3%) patients on abdominal ultrasound. There were no significant differences in frequency of hospital admissions, causes of admission, blood transfusions, or malaria episodes when comparing individuals with and without splenomegaly. However, patients with splenomegaly (n=9) had significantly higher levels of hemoglobin (P=0.02), haematocrit (P=0.003), and fetal haemoglobin (P=0.01). In addition, white blood cell counts (P=<0.001), platelets (P=<0.001), mean corpuscular hemoglobin (P=0.03) and mean corpuscular hemoglobin concentration (P=<0.001) were significantly lower in those with splenomegaly (Table 1). There were no differences in the levels of markers of hemolysis. Cholelithiasis was observed in 38 (20.5%) patients among those with no palpable splenomegaly and in only 1 (11.1%) patient out of those with splenomegaly (P=0.69). Howell-Jolly bodies were present in thin smears of all patients, with no significant difference between the two groups.Conclusion:Persistent splenomegaly exists in SCA patients in Tanzania and is correlated with laboratory markers that signify less severe disease. Future work, including a matched case control study and assessment of splenic function, will help to identify causes of splenomegaly in patients with SCA and to determine if splenomegaly is a predictor of disease severity in SCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genotypic Diversity among Angolan Children with Sickle Cell Anemia.

Background. Sickle cell anemia (SCA) is an inherited blood disorder that affects over 300,000 newborns worldwide every year, being particularly prevalent in Sub-Saharan Africa. Despite being a monogenic disease, SCA shows a remarkably high clinical heterogeneity. Several studies have already demonstrated the existence of some polymorphisms that can provide major clinical benefits, producing a mild phenotype. Moreover, the existence of distinct haplotypes can also influence the phenotype patterns of certain populations, leading to different clinical manifestations. Our aim was to assess the association between polymorphisms in genes previously related to SCA disease severity in an Angolan pediatric population. Methods. This study analyzed clinical and biological data collected from 192 Angolan children. Using NGS data, we classified the HBB haplotypes based on four previously described SNPs (rs3834466, rs28440105, rs10128556, and rs968857) and the genotype for the SNPs in HBG2 (rs7482144), BCL11A (rs4671393, rs11886868, rs1427407, rs7557939), HBS1L-MYB (rs66650371) and BGLT3 (rs7924684) genes. Results. The CAR haplotype was undoubtedly the most common HBB haplotype in our population. The HbF values and the ratio of gamma chains were statistically significant for almost all of the variants studied. We reported for the first time an association between rs7924684 in the BGLT3 gene and gamma chains ratio. Conclusions. The current findings emphasize the importance personalized medicine would have if applied to SCA patient care, since some of the variants studied might predict the phenotype and the overall response to treatment.

The Impact of Glucose-6-Phosphate Dehydrogenase Deficiency on the Frequency of Vasoocclusive Crisis in Patients with Sickle Cell Anemia

Background and ObjectivesSickle cell anemia (SCA) and glucose-6-phosphate dehydrogenase (G6PD) deficiency are both hereditary diseases of the red blood cells that cause hemolysis. The impact of the interaction of both conditions on the clinical and laboratory presentations of the affected persons is sparse. This study, therefore, correlated G6PD activity with disease severity in persons with SCA by comparing disease severity in G6PD-deficient SCA persons with those with normal G6PD activity.MethodologyThis cross-sectional study was conducted in the department of Haematology and Blood Transfusion of the Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria. G6PD activity, SCA disease severity, and hematological parameters including reticulocyte counts and Heinz body estimation, bilirubin, and aspartate transaminase were estimated in 67 SCA persons. The results were compared between SCA persons with G6PD deficiency and those with normal enzyme activity.ResultsThe prevalence of G6PD deficiency was found to be 23.9%. The G6PD-deficient SCA patients included 4 (25.0%) males and 12 (75.0%) females. G6PD deficiency was significantly higher in females ( P = .047). There was no significant difference in disease severity scores between G6PD-deficient and G6PD-nondeficient SCA patients. However, G6PD-deficient persons reported significantly higher episodes of severe vasoocclusive crisis (VOC) per annum ( P = .048). The hematological and biochemical parameters were similar between G6PD-deficient and G6PD normal SCA persons except that the G6PD-deficient SCA persons have significantly higher reticulocyte response ( P = .001). There was no correlation between disease severity resulting from reduced G6PD activity and Heinz body formation in SCA persons in the steady state.ConclusionG6PD deficiency significantly contributes to recurrent painful vasoocclusive crisis in SCA persons in the steady state.

Red Blood Cell Deformability Demonstrated with the Oxygenscan: Exploring the Association with Hydroxyurea Treatment, Co-Inherited α-Thalassemia, and Frequency of Pain in Children with Sickle Cell Anemia

Background Sickle hemoglobin (hemoglobin S, HbS) is a structural variant of adult hemoglobin. HbS polymerizes when oxygen tensions are low, leading to red blood cell (RBC) deformation, so-called "sickling". In sickle cell anemia (SCA), loss of RBC deformability is considered to be a primary factor responsible for vaso-occlusion and hemolysis. Until recently no laboratory tests to measure RBC deformability in SCA have been readily available. Study Aims In this study we examine RBC deformability, measured with the oxygenscan module of the Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer, in children with SCA treated with or without hydroxyurea (HU). Furthermore, we investigate the relationship between RBC deformability and pain frequency, as well as genetic and laboratory measures known to be associated with disease severity in SCA. Methods We included children aged 0-16 years with a confirmed diagnosis of SCA (HbSS) from the pediatric sickle cell clinic at King's College Hospital in London. Children were excluded if they had received any blood transfusions within 3 months of study inclusion. Children on HU were only included if treatment had been initiated &gt;3 months prior to recruitment and the dose was stable. Children and their parents or guardians reported frequency of pain as: daily, weekly, monthly, yearly, or never. Laboratory measurements, including total hemoglobin (hb), hemoglobin F (HbF), and reticulocyte percentage, were performed on the same day as a sample was taken for oxygenscan analysis. Data on co-inheritance of α-thalassemia was recorded if available. EDTA blood samples were kept at approximately 4°C and transported from King's College London to Copenhagen University Hospital (Herlev and Gentofte Hospital), where they were analyzed within 48 hours of sampling using the Lorrca oxygenscan (RR Mechatronics, the Netherlands). The oxygenscan measures RBC deformability expressed as an elongation index (EI) during deoxygenation and reoxygenation, with EImax expressing RBC deformability at normal oxygen concentrations, EImin expressing RBC deformability after deoxygenation, and the point of sickling (POS) expressing the point at which &gt;5% decrease in EI is observed, representing the pO2 at which sickling begins. All statistical analyses were performed in Stata V16.0 (StataCorp. 2019, USA), using the two-sided t-test, one-way ANOVA, and Pearson's correlation when appropriate. Results We included 47 children aged 0-16 years (mean age 7.9 years) in the study, 24 (51%) receiving HU. Children in the HU group presented with significantly higher HbF percentage compared to the non-HU group (15.6% and 10.9%, p=0.03). Children receiving HU had higher EImax and EImin, and lower POS values, compared to children in the non-HU group, although results were not significant (Table 1). There was a positive correlation between HbF and EImax (r= 0.57, p=0.0001) and HbF and EImin (r= 0.56, p=0.0001), and a negative correlation between HbF and POS (r=-0.37, p=0.01), as well as a positive correlation between total hb and EImax (r=0.35, p=0.02). There was no significant correlation between any oxygenscan parameters and reticulocyte percentage. Data on α-thalassemia was available for 23 children. EImax and EImin values were higher in heterozygous children compared to children without co-inherited α-thalassemia, and POS values were lower, but results were not significant (Table 2). We found no significant association between any oxygenscan parameters and pain frequency (Table 3). Conclusion In this study we identified a strong correlation between all oxygenscan parameters and HbF percentage, as has been reported previously. We found higher EImax and EImin and lower POS values in children receiving HU treatment and children with co-inherited heterozygous α-thalassemia, suggesting increased RBC deformability in these children. These results were not significant, however, which may in part be due to lack of power in the study. Also, it is possible that children in the HU group would have presented with lower EImax and EImin and higher POS values prior to HU initiation, with treatment response leading to results similar to those found in the non-HU group. Finally, our results suggest that there is no association between oxygenscan parameters and self-reported frequency of pain in children with SCA. Disclosures No relevant conflicts of interest to declare.

The Oxygenscan: A Rapid and Reproducible Test to Determine Patient-Specific, Clinically Relevant Biomarkers of Disease Severity in Sickle Cell Anemia

Background: In sickle cell anemia (SCA), hemoglobin S (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These deoxygenated RBCs have strongly reduced deformability, which contributes to the etiology of vaso-occlusive crises and chronic hemolytic anemia. There are no widely available clinical laboratory tests to directly monitor effects of disease modifying therapies (i.e. hydroxyurea) on RBC deformability. RBC deformability can be measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, the Netherlands), which measures RBC deformability over a range of osmolalities. Recently, a new module was added which consists of a method to measure RBC deformability, expressed as Elongation Index (EI), during controlled deoxygenation. This test, termed oxygenscan, has 3 key read out parameters: 1) EImax, which represents RBC deformability at normoxia; 2) EImin represents deformability upon deoxygenation; and 3) the point of sickling (PoS), the point at which a >5% decrease in EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1). In this study, we correlated laboratory parameters associated with SCA disease severity with oxygenscan parameters to establish the clinical utility of this test.Methods: The discovery cohort consisted of 15 SCA patients (median age 22.0 years, 33.3% on hydroxyurea (HU)) enrolled at University Medical Center Utrecht (UMCU). The validation cohort consisted of 21 patients with SCA (median age 12.5 years, 76.2% on HU) from Texas Children's Hematology Center (TCHC). Oxygenscans were carried out in duplicates at both sites. Percentage dense RBC (%DRBC) were measured using an ADVIA hematology analyzer (Siemens) at TCHC only. In this study, we used Pearson's correlation to test for linear correlations between oxygenscan parameters EImax, EImin and PoS and clinically relevant laboratory parameters: total hemoglobin (Hb), absolute reticulocyte count (ARC), %HbS and %HbF, and %DRBC.Results: In both cohorts PoS significantly positively correlated with ARC (Figure 2A-B). In the UMCU cohort, total Hb levels also significantly positively correlated with EImax (Figure 2C), which was validated in the TCHC cohort (Figure 2D). HbF positively correlated with the EImin in both cohorts (Figure 2E-F). EImin also significantly negatively correlated with HbS (r=-0.828 p=<0.001 in the UMCU cohort, r=-0.936, p=<0.001 in the TCHC cohort data not shown). EImax showed a strong negative correlation with the %DRBC (Figure 2G) in the TCHC cohort. Individual test results were highly reproducible at both sites, with a median coefficient of variability of all tested parameters below 3%.Conclusion: The oxygenscan is a semi-automated, inexpensive, highly reproducible, and rapid test to fully characterize patient-specific RBC deformability under a range of oxygen concentrations. Key oxygenscan measurements- PoS, EImin, and EImax- correlated with known measures of SCA disease severity, namely ARC, HbF, HbS, total Hb and %DRBC. Patients with higher reticulocyte counts showed a clinically unfavorable increase of oxygen concentration at which RBCs start to sickle (termed PoS), than patients with lower ARC. Patients with higher HbF had more deformable RBCs even at the lowest oxygen concentrations, or EImin, while patients with higher HbS had lower EImin (low values indicate poor deformability under deoxygenated conditions). Patients with high %DRBC had lower EImax, indicating poor RBC deformability at normoxic conditions. Conversely, patients with high total Hb had high EImax.The very strong correlations of key oxygenscan measurements with different measures of SCA disease severity suggest that these parameters could be exploited as useful biomarkers of clinical severity and in the follow-up and treatment of SCA patients and warrant further investigation. [Display omitted] DisclosuresRab:RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Cnossen:Roche: Research Funding; CSL Behring: Research Funding; Novartis: Research Funding; Novo Nordisk: Research Funding; Bayer: Research Funding; Shire: Research Funding; Pfizer: Research Funding. Schutgens:Bayer: Research Funding; CSL Behring: Research Funding; Pfizer: Research Funding; Uniqure BV: Research Funding; Novo Nordisk: Research Funding; Baxalta/Shire: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding. van Beers:RR Mechatronics: Research Funding; Bayer: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The role of BCL11A and HMIP-2 polymorphisms on endogenous and hydroxyurea induced levels of fetal hemoglobin in sickle cell anemia patients from southern Brazil

High levels of fetal hemoglobin (HbF) reduce sickle cell anemia (SCA) morbidity and mortality. HbF levels vary considerably and there is a strong genetic component that influences HbF production. Genetic polymorphisms at three quantitative trait loci (QTL): Xmn1-HBG2, HMIP-2 and BCL11A, have been shown to influence HbF levels and disease severity in SCA. Hydroxyurea (HU) is a drug that increases HbF. We investigated the influence of single nucleotide polymorphisms (SNPs) at the Xmn1-HBG2 (rs7482144); BCL11A (rs1427407, rs4671393 and rs11886868); and HMIP-2 (rs9399137 and rs9402686) loci on baseline and HU-induced HbF levels in 111 HbSS patients. We found that both BCL11A and HMIP-2 were associated with increased endogenous levels of HbF. Interestingly, we also found that BCL11A was associated with higher induction of HbF with HU. This effect was independent of the effect of BCL11A on baseline HbF levels. Additional studies will be needed to validate these findings and explain the ample inter-individual variations in HbF levels at baseline and HU-induced in patients with SCA.

Genetic Modifiers of White Blood Cell Count, Albuminuria and Glomerular Filtration Rate in Children with Sickle Cell Anemia.

Discovery and validation of genetic variants that influence disease severity in children with sickle cell anemia (SCA) could lead to early identification of high-risk patients, better screening strategies, and intervention with targeted and preventive therapy. We hypothesized that newly identified genetic risk factors for the general African American population could also impact laboratory biomarkers known to contribute to the clinical disease expression of SCA, including variants influencing the white blood cell count and the development of albuminuria and abnormal glomerular filtration rate. We first investigated candidate genetic polymorphisms in well-characterized SCA pediatric cohorts from three prospective NHLBI-supported clinical trials: HUSTLE, SWiTCH, and TWiTCH. We also performed whole exome sequencing to identify novel genetic variants, using both a discovery and a validation cohort. Among candidate genes, DARC rs2814778 polymorphism regulating Duffy antigen expression had a clear influence with significantly increased WBC and neutrophil counts, but did not affect the maximum tolerated dose of hydroxyurea therapy. The APOL1 G1 polymorphism, an identified risk factor for non-diabetic renal disease, was associated with albuminuria. Whole exome sequencing discovered several novel variants that maintained significance in the validation cohorts, including ZFHX4 polymorphisms affecting both the leukocyte and neutrophil counts, as well as AGGF1, CYP4B1, CUBN, TOR2A, PKD1L2, and CD163 variants affecting the glomerular filtration rate. The identification of robust, reliable, and reproducible genetic markers for disease severity in SCA remains elusive, but new genetic variants provide avenues for further validation and investigation.

Plasma lactate dehydrogenase level as indicator of severe Homozygous sickle cell disease

Background: Homozygous sickle cell disease (SCD) (SS), also called sickle cell anemia (SCA) is the most common SCD in Nigeria. Studies done in the developed world showed that serum lactate dehydrogenase (LDH) levels correlated positively and significantly with clinical severity of the disease. This study attempts to provide information on the relationship between the plasma LDH level and the clinical severity of SCA in Nigerian children. Objective: The objective of this work was to assess plasma LDH level in Nigerian SCA children, aged 5-15 years, as a clinical indicator of disease severity. Methods: Plasma LDH level was measured quantitatively using Randox LDH reagent kit. A semi-quantitative assessment of clinical severity of SCA was carried out on all the SCA subjects using the Bienzle et al. assessment profile modified for this study. Subjects were classified as having mild, moderate, or severe disease based on their clinical severity score. Data were analyzed using Statistical Package for Social Sciences (SPSS) version 21 software. Results: Eighty SCA children, 40 in steady state and 40 in hemolytic (with or without vaso-occlusive) crises were studied. Mean plasma LDH level was significantly higher in SCA subjects in crises than for those in steady state (P = 0.0284). Only steady state plasma LDH level correlated positively and significantly with clinical severity score (P = 0.0151). Conclusion: Plasma LDH appears to be a reliable indicator of SCA disease severity in the steady state in Nigerian children.

Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study

BackgroundFrequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.MethodsTwo hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.ResultsHigher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality. ConclusionsSevere painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.Trial RegistrationClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/

Gallstones in a Newborn-Cohort with Sickle Cell Anemia (SCA): Cumulative Risk and Predictive Factors

Abstract 513 Background:The accelerated hemolysis in SCA increases bilirubin excretion and the susceptibility to pigment gallstone formation. Uridine diphosphate-glucuronosyltransferase (UGT1A1) gene polymorphism and alpha-thalassemia are factors known to modify this risk. However, their relative influence and other factors such as SCA disease severity and the use of ceftriaxone for acute febrile episodes remain to be explored in a newborn cohort. The goal of this study was to define the cumulative incidence and risk factors for the occurrence of gallstones in a SCA-newborn-cohort. Patients and Methods:Alpha-genes, beta-globin haplotypes, UGT1AI genotype, G6PD-enzymatic activity were recorded; baseline biological parameters were determined during the second year of life, away from crises and transfusions as previously described (Blood 2011,117, 4). Annual check-ups including abdominal sonography were performed as soon as the age of 1 year. Cholecystectomy was systematically performed in the 6 months following the discovery and confirmation of presence of gallstones. All hospitalizations and their causes were prospectively recorded. Patients were censored on the date of gallstone occurrence or last abdominal sonography; event rates/year (pain crises, ACS, febrile episodes) were calculated, and KM-estimated cumulative risk for gallstones and Cox regression were used to assess the predictive factors for gallstones. Results:SCA-patients (364: 353 SS, 8 Sb0, 3 SDPunjab; 182 F,182 M) born before February 2010 and followed until the age of 18–20 years were included in this study. Alpha-thalassemia was present in 46% patients (available in 323; 2 genes: n=33; 3 genes: n=116; 4 genes: n=172; 5 genes: n=2); beta-haplotypes (available in 291) were Car/Car in 42.6%, Ben/Ben in 19.9%, Sen/Sen in 9.6%, and others in 27.8%. G6PD activity (available in 309) was deficient in 11.6%. UGTA1 polymorphism (available in 175) showed Gilbert genotype in 19.8%, i.e., 7/7 in 13.4% and 7/8 in 6.4%. The frequency of the other genotypes was: 5/6: 6.4%; 5/7:7%; 5/8:0.6%; 6/6:23.3%, 6/7:34.3%; and 6/8:8.1%. Unconjugated bilirubin values at baseline were significantly correlated with the total number of UGTA1A1 repeats (r=0.415, p<0.001). Mean rates (SD) of pain crises, ACS and acute febrile episodes were 0.56 (0.68), 0.12 (0.18), 0.34 (0.54), respectively. Gallstones were observed in 98/364 patients at the median age of 9.2 (range: 2.5 to 17.9 years). The cumulative risk of gallstones was 5% by age 5, 20.7% by age 10 and 35.6 % by age 15 and was not significantly different in patients with Gilbert polymorphism (Log Rank p=0.4) but was significantly related to the number of alpha-genes (Log-Rank p=0.039). However, the risk was significantly increased in patients homozygous or heterozygous for (TA8) (Log Rank p=0.016). Univariate Cox regression analysis showed that the number of alpha-genes was a significant risk factor (p=0.014) whereas G6PD deficiency, beta-haplotypes and Gilbert genotype were not significant risk factors; however, the presence of at least one allele (TA8) was a significant risk factor (p=0.025). Among the baseline biological parameters, hemoglobin, WBC, neutrophils, platelets, MCV, and bilirubin were not significant factors whereas HbF level (p=0.028), reticulocyte count (p<0.001) and LDH (p=0.020) significantly increased the risk for gallstones. Among clinical events, pain crises, ACS and febrile episodes significantly increased the risk (p<0.001). Multivariate Cox analysis including all the significant risk factors in the univariate analysis retained the deletion of 2 alpha genes (HR=4.66; 95% CI:1.11-19.52,p=0.035) which decreases the risk, the presence of at least one allele (TA8) (HR=2.26:95% CI:1.07-4.78, p=0.032), which increases the risk and the baseline reticulocytes count per 1×109/L increase(HR:1.001;1.000-1.002;p=0.005), as independent and significant predictive factors for gallstones. Conclusion:This prospective SCA newborn-cohort study shows that gallstones may begin as soon as 2.5 years of age and that the cumulative risk reaches a high 35.6% by age 15. The deletion of 2 alpha genes, the presence of at least one UGT1A1 (TA8) allele and high baseline reticulocyte count are independent and significant predictive factors showing that the risks are multifacturial and highly and primarily dependent on hemolysis [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Pomalidomide Modifies Sickle Cell Related Organ Damage in Transgenic Mice with Sickle Cell Anemia.

Abstract 904 Introduction:We previously reported that Pomalidomide (PL), a novel thalidomide-derived immunomodulatory drug (IMiD), is capable of enhancing erythropoiesis and fetal hemoglobin (HbF) production in a knockout-transgenic (KT) mouse model of sickle cell anemia (SCA). In addition to these hematological properties, PL is known to modulate specific effector functions of the innate and adaptive immune system. PL and other IMiDs potently inhibit the output of the inflammatory mediators TNF-α, IL-1β, and IL-6 in activated monocytes/macrophages, whereas their ability to promote T helper 1 (Th1) lymphocyte differentiation and co-stimulation of both CD4+ and CD8+ T lymphocytes can lead to increased TNF-α, IL-2, and IFN-γ secretion in both cell culture and human subjects. SCA is an inflammatory disease. It is therefore conceivable that PL could diminish disease severity in SCA by targeting vascular endothelial and innate immune cell activation, whereas preferential induction of a Th1-biased lymphocyte program could have unwanted effects. To test these possibilities in SCA, we evaluated PL's immunomodulatory activities in a relevant KT mouse model. Methods:Animals. Six week old KT homozygous sickle mice were treated daily (Mon-Fri; i.p. injections) for eight weeks with Vehicle (n=8) or PL (10 mg/kg; n=9). Mice were maintained in an accredited pathogen-free animal facility according to NIH and institutional guidelines. Mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture into 0.5 ml vacutainer EDTA tubes (Becton-Dickinson). Soluble plasma adhesion molecules (sVCAM-1, sICAM-1, and sE-Selectin) and cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α, G-CSF, and GM-CSF) were measured by ELISA (R&D Systems and SABiosciences). Organ analysis. We focused our analysis on the liver of KT mice because this organ manifests severe sickle cell-related pathology. Livers were removed and divided for H&E paraffin sections, immunohistochemistry, and RNA analysis (storage at -80°C). The area of liver ischemic infarcts (ALI) was measured on H&E tissue sections using an image processing program (Image J, NIH) and averaged from four randomly selected low power optical fields (5x) / animal. Whole liver RNA was pooled from PL animals with low ALI scores (n=2) and vehicle animals (n=3) using Trizol followed by RNeasy column purification. RNA was submitted to SABioscience for analysis using the 440 gene Inflammatory Response and Autoimmunity GEArray. Statistical analysis. One-Way ANOVA/Student-Newman-Kuels and Kruskal-Wallis One-Way ANOVA/Dunn's Method (Sigma Stat). Data are reported as the mean ± SE. A P-value of < 0.05 was considered significant. Results:The peritoneal cavity of all animals was free of adhesions, exudates and drug, suggesting that daily i.p. injections did not inflame the peritoneal membranes and resulted in complete absorption of the active compound. PL significantly reduced the level of the endothelial cell marker of inflammation, sVCAM-1 (sVCAM-1[ng/ml]: Veh: 1202±36; PL: 962±48; P<0.01), but did not affect sICAM-1 or sE-Selectin. Plasma cytokines in vehicle animals were measured at the assay's lower limit of detection and were not significantly modified by PL, suggesting that PL did not induce overt activation of the TH1 lymphocyte program. Liver histology in vehicle controls revealed scattered tissue infarcts surrounded by a mixed inflammatory cell infiltrate (macrophages [F4/80+], T-lymphocytes [CD3+]). PL reduced the ALI by 60 % (ALI [mm2/LPF: Veh: 0.30±0.05; PL: 0.12±0.06; P=0.01). The ALI score of eight out of nine PL mice was lower than the lowest score in the vehicle group (89% responder rate). Gene array data were consistent with decreased organ inflammation (serum amyloid A2 [-3.57 fold]), modifications in antigen presentation (CD74 [-2.12 fold]), inhibition of T-cell signaling (SLAP-2 [+5.68 fold]; SLP-76 [-1.92 fold]), and reduced T-cell migration (MIP-3β [-2.56 fold]). Tissue protection in the PL group did not correlate with HbF expression, total WBC count, or any other hematological variable. Summary & Conclusions:Pomalidomide modulates vascular and tissue markers of inflammation and protects from sickle cell-induced organ damage in a HbF-independent manner. These data suggest that PL, in addition to its HbF-inducing properties, may exert beneficial anti-inflammatory effects in SCA. Disclosures:Meiler:Celgene: Research Funding. Wade:Celgene: Research Funding. Moutouh de Parseval:Celgene: Employment. Corral:Celgene: Employment. Swerdlow:Celgene: Research Funding. Kutlar:Celgene: Research Funding.

Active Von Willebrand Factor (VWF) in Plasma of Sickle Cell Patients Is An Indicator of Disease Severity.

Abstract 2552Poster Board II-529Vasoocclusion, hemolysis and oxidative stress are hallmarks of sickle cell anemia (SCA). Oxidants generated in SCA can stimulate endothelial cell release of ultra-large VWF (ULVWF), which is hyperactive in binding platelets and erythrocytes compared to normal plasma VWF, and binds sickle erythrocytes especially well. VWF levels in plasma as assessed by standard ELISA (VWF:Ag) have consistently been shown to be increased in patients with SCA; however, the extent of increase has not been demonstrated to correlate with disease severity. Given VWF's role as an adhesive ligand for several types of blood cells, including erythrocytes, we hypothesized that its contribution to SCA disease severity and pathophysiology would be better evaluated by measuring both its plasma concentration and its adhesive activity. We assessed several parameters of VWF quantity and function in plasma from SCA patients at disease baseline (n=13 including 9 homozygous sickle cell disease, 2 sickle-β+ thalassemia, 1 sickle-β0 thalassemia, and 1 hemoglobin SC ). Parameters included VWF:Ag, multimer pattern, ADAMTS13 activity, ADAMTS13 antigen, and VWF Activation Factor (VWF-AF). VWF-AF was determined using a llama-derived antibody fragment (nanobody) that detects an active conformation of the VWF A1 domain correlating with enhanced platelet binding. Elevated VWF-AF has been demonstrated in patients with type 2B von Willebrand disease, thrombotic thrombocytopenic purpura, HELLP syndrome, and other thrombotic disorders in which ULVWF is increased. VWF-AF is calculated as the ratio of nanobody binding in patient plasma to that of normal pooled plasma (NPP) at a given VWF:Ag level (the activation factor of NPP is set at 1). We also derived a novel, calculated measure of VWF reactivity present in plasma, termed Total Active VWF (TA-VWF), by multiplying the VWF-AF by VWF:Ag for each patient sample. As expected, all SCA patients had VWF:Ag levels that were higher than in NPP (1.2–3.6 fold) or in ethnically matched control plasma. VWF-AF was elevated in 9/13 individuals (0.6 to 4.4 fold). TA-VWF was increased in 11/13 patients (0.7 to 10.1 fold), which correlated with an increased size of VWF multimers on agarose gels. Individuals with the lowest VWF-AF and TA-VWF were those with genotypes consistent with less severe disease (sickle-β+ thalassemia and Hemoglobin SC). Among individuals with homozygous SS disease and sickle-β0 thalassemia, those with more severe clinical disease had higher TA-VWF than those with mild disease. Furthermore, TA-VWF significantly correlated with plasma lactate dehydrogenase (LDH) (P = 0.007), a measure of hemolysis that is associated with increased risk of pulmonary artery hypertension, leg ulcers, priapism, and sudden death in SCA. Consistent with this finding, TA-VWF also correlated (P < 0.01) with a non-invasive measures of pulmonary artery hypertension, the triscuspid regurgitent jet velocity. Increased VWF-AF was not correlated with increased VWF:Ag. We did not detect deficiency of ADAMTS13 activity using a sensitive assay measuring the ability of plasma ADAMTS13 to cleave a recombinant VWF A2 domain peptide substrate in vitro under static conditions. ADAMTS13 antigen in our patient population was similar to NPP. These findings suggest that elevated levels of active VWF predispose patients with SCA to disease complications. The increased VWF reactivity likely arises from increased endothelial secretion of ULVWF and activation of the VWF A1 domain in plasma of SCA patients. This also suggests that VWF reactivity is a pathophysiologic link between adhesion and hemolysis in SCA, and likely represents a novel therapeutic target. Disclosures:No relevant conflicts of interest to declare.