Sickle Cell Anemia Disease Research Articles

5554869 ASSOCIATION BETWEEN HEMOGLOBIN LEVELS AND END-ORGAN DAMAGE IN SICKLE CELL DISEASE: A RETROSPECTIVE LINKED PRIMARY AND SECONDARY CARE DATABASE ANALYSIS IN ENGLAND

Background: Sickle cell disease (SCD) is a chronic, multifaceted blood disorder that affects the oxygen-carrying protein hemoglobin (Hb). Along with chronic hemolysis and vaso-occlusion that often accompanies SCD, low Hb levels may contribute to downstream end-organ damage (EOD). Emerging evidence suggests an association between chronic hemolytic anemia in SCD and both short-term and long-term adverse outcomes, including EOD. Therefore, treatments that directly increase Hb may improve patients’ acute symptoms while reducing the risk for long-term disease-related effects known to contribute to morbidity and mortality. Aims: This study aims to ascertain the association between Hb level variation and EOD and clinical conditions typically associated with SCD (stroke, pulmonary hypertension, chronic kidney disease [CKD], end-stage renal disease [ESRD], leg ulcers, and pneumonia) in a real-world data set. Methods: The Clinical Practice Research Datalink (CPRD) and the Hospital Episode Statistics (HES) databases in England were used to retrospectively identify linked Hb data among patients with SCD (April 1, 2007, to March 31, 2019). Recurring results in any 90-day period were averaged. Logistic regression adjusted with demographic and clinical variables was used to determine odds ratios (ORs) of developing each EOD outcome or clinical complication. ORs, P values, 95% confidence intervals (CIs), and areas under the receiver operating curve (AUC-ROCs) and corresponding 95% CIs were reported. Results: Of 11,936 patients identified, 5379 (45%) had Hb results recorded in the CPRD. The logistic model for risk assessment of all outcomes studied indicated that an increase in Hb among patients with SCD was associated with a statistically significant (P<0.001) decrease in adjusted ORs per 1 g/dL increase in Hb (Table). Except for ESRD, leg ulcers, and pneumonia, the adjusted models showed statistically significant increases in AUC-ROC compared with the unadjusted models, signifying that the adjusted models were more robust and further verified a statistical OR after adjustment. Increased Hb was associated with decreased risk of respiratory-related events. Pulmonary hypertension had the strongest effect with the most robust model (ORadjusted of 0.66). For pneumonia, ORadjusted was 0.77. For renal insufficiency, ORs for developing CKD or ESRD were significantly lower than 1 (ORadjusted=0.73 and ORadjusted=0.75, respectively). After adjusting for clinical and demographic factors, the model predicted a lower risk for stroke (ORadjusted=0.89). The estimated AUC-ROCs suggest that the adjusted model is more robust in stroke, hypertension, and CKD. These findings did not change when testing for other covariates such as deep vein thrombosis. Summary-Conclusion: Among patients with SCD, an increase in Hb of 1 g/dL was associated with a statistically significant reduction in risk for 6 common EOD outcomes and clinical conditions. The results were more pronounced after adjusting for demographic and clinical covariates (based on clinical opinion and literature review) and further assessed with a bivariate analysis. The results were obtained from a representative real-world evidence dataset analyzed over a 12-year period, which was sufficient to observe EOD events, and are generalizable to the UK and similar populations. Our findings support the use of therapeutics that increase Hb in patients with SCD to protect against deleterious organ damage.

5613396 DIFFERENCE IN HOSPITALIZATIONS AND ANNUAL BED DAYS FOR PATIENTS WITH SICKLE CELL DISEASE BY VARYING LEVELS OF ANEMIA: A RETROSPECTIVE ANALYSIS OF LINKED PRIMARY AND SECONDARY CARE DATABASES IN ENGLAND

Background: Sickle cell disease (SCD) is a life-threatening autosomal-recessive disorder in which the sickle variant of hemoglobin (Hb) polymerizes under deoxygenation, causing red blood cell sickling and hemolytic anemia. Anemia in SCD is associated with an increased risk of complications such as leg ulcers and end-organ damage (EOD), which often require hospitalization, a key driver of healthcare costs. Aims: To quantify differences in all-cause hospitalizations and annual bed days in patients with SCD and differing levels of anemia. Methods: We identified 6018 patients with SCD aged ≥12 years with ≥1 recorded Hb level between January 1, 2007, and March 31, 2019, from the Clinical Practice Research Datalink (Aurum version), an English longitudinal primary care electronic medical record, and linked these with Hospital Episode Statistics, a secondary care reimbursement data set. We obtained Hb levels from primary care records, when disease is expected to be relatively stable, as opposed to during hospitalization. We used Hb measurements separated by <30 days to calculate mean Hb for a time interval at a specific Hb level. Patients were grouped into Hb categories based on having ≥1 Hb record of <6 g/dL (severe anemia), 6 to <8 g/dL, 8 to 10.5 g/dL, or >10.5 g/dL; patient Hb category may have changed over the course of the study. Patient Hb intervals began with Hb in a category and ended on the day before a blood transfusion, with a Hb reading in a new category, or on the last activity date. We compared the significance of the categorical data distribution for hospitalizations using the Kruskal–Wallis test across Hb categories, extended with pairwise Wilcoxon rank sum testing. We repeated the above for hospitalizations due to any EOD and annual bed days. Results: Of 6018 patients with recorded Hb, 72% had a history of Hb >10.5 g/dL, 44% had 8 to 10.5 g/dL, 15% had 6 to 8 g/dL, and 3% had <6 g/dL. The annual rate of all-cause hospitalizations decreased as Hb increased: 5.84 in patients with Hb <6 g/dL, 4.13 for 6 to 8 g/dL, 3.3 for 8 to 10.5 g/dL, and 1.7 for >10.5 g/dL. Hospitalizations were significantly lower for patients with a history of Hb >10.5 g/dL than for those with Hb <6 g/dL (P<0.001); across Hb categories, the distribution of all-cause hospitalization was significant (P<0.001). Annual all-cause bed days decreased across increasing Hb categories, with a mean of 8.8 days in patients with a history of Hb <6 g/dL, 7.7 days with 6 to 8 g/dL, 5.8 days with 8 to 10.5 g/dL, and 3.2 days with >10.5 g/dL. A significant decrease in annual bed days was observed in patients with a history of Hb >10.5 g/dL compared with Hb <6 g/dL (P<0.001). Across Hb categories, the distribution of the difference in bed days was significant (P<0.001) (Figure).A total of 385 patients (6.4%) had ≥1 hospitalization for any EOD. Significantly fewer hospitalizations were attributable to any EOD in patients with a history of Hb >10.5 g/dL compared with those with a history of Hb <6 g/dL (0.36 and 2.4 hospitalizations per patient interval per year, respectively; P<0.001). Summary/Conclusion: All-cause hospitalizations and annual bed days were progressively reduced in patients with a history of higher Hb levels whereas patients with severe anemia did not experience the same benefit. Due to this tendency, appropriate measures should be taken to maximize Hb in patients with SCD. Reference 1. Kato GJ, Piel FB, Reid CD, et al. Sickle cell disease. Nat Rev Dis Primers. 2018;4:18010.

Disease severity and renal function among sickle cell anaemia patients in a tertiary hospital, South-south, Nigeria: a cross sectional study.

Renal disease is a recognized complication of sickle cell anaemia (SCA), especially from the third decade of life and is linked to disease severity. This study assessed the association between disease severity and renal function among SCA patients using routine and newer markers of renal function. This cross-sectional study recruited 85 SCA patients. Disease severity was assessed using modified Adegoke criteria which include the frequency of transfusion, painful crises, packed cell volume, and history of complications such as hypertension and chronic leg ulcers. Renal function was assessed using urea, creatinine, and beta-2-microglobulin (β2-M). Association was determined between renal function and disease severity using Pearson's correlation. P-value < 0.05 was taken as significant. The mean age of participants was 27.2 ± 7.6 years with 41(48.2%) males and 44 (51.8%) females. The mean packed cell volume, serum creatinine, serum urea, and β2-M were 24.0± 4.1%,17.6±7.5 mg/dL, 0.7±0.3mg/dL, 3.4±1.2mg/l respectively. A majority (54.1%) of them had a mild disease while 35.3% and 10.6% had moderate and severe diseases, respectively. Forty of the SCA patients had urine specific gravity below 1.010. The mean values of systolic blood pressure (p=0.001) diastolic blood pressure (p=0.001), serum creatinine (p=0.028) and β2M (p=0.019) significantly increased with disease severity. There was a significant positive correlation between SCA disease severity and serum urea (r=0.229; p=0.035), and serum β2-microglobulin (r=0.270; p=0.012). Sickle cell anaemia severity is associated with a decline in renal function using both traditional and novel renal markers. Serum β2-M may serve as a useful marker of renal function and disease severity in SCA.

5613510 EFFECTS OF REGIONAL BRAIN VOLUME ON COGNITION IN SICKLE CELL ANAEMIA: A DEVELOPMENTAL PERSPECTIVE

Cognitive decline, related at least in part to reduced processing speed and lower working memory, is a major problem in paediatric and adult patients with sickle cell anaemia (SCA)1,2 and affects quality of life. Multiple studies investigating the association between quantitative and qualitative neuroimaging findings and cognition have had mixed results. Hence, the aetiology of cognitive decline in this population is not clearly understood. Cerebral atrophy on magnetic resonance imaging is noted in the literature but few studies have investigated the association between brain volumes and cognition. White matter microstructural integrity and white matter hyperintensities have been associated with reduced processing speed index (PSI).3 However no study has investigated the association between white matter volumes and PSI in the SCA population. Total subcortical volumes have also been noted to be lower in SCA children and adults. Only one study has investigated the association between total subcortical volumes and working memory index (WMI) in adults.4 Moreover, several studies in the past have noted age related decline in total brain volume as well neurodevelopmental delay in grey matter volume growth.5,6 Most of the developmental trajectory data in the literature comes from longitudinal data sets with limited samples and without control groups. Hence, for the purpose of this study, we investigated the association between regional brain volumes (white matter volumes and total subcortical volumes) and cognition (processing speed index and working memory index) in 129 SCA patients aged 8 – 64 years (66 male) and 50 controls (21 males). Participants were enrolled in two studies - the Sleep Asthma Cohort (SAC) and the Prevention of Morbidity in SCA (POMS) trial. We hypothesized that white matter volumes would predict processing speed index and total subcortical volumes would predict working memory index in SCA patients as well as controls. We also hypothesized that the developmental trajectories for regional brain volumes and cognitive variables would differ between patients and controls. Separate multiple linear regression models for males and females revealed that WMV predicted processing speed index(p=.019) and total subcortical volumes predicted working memory index (p=.025) in male patients only. Age (p=.003) and socioeconomic status (p=.03) also independently predicted PSI in male patients. Individual subcortical regions predicting WMI were statistically significant for left and right thalamus only in male patients. Haemoglobin and age emerged as the best predictors of cognitive decline in patients. Comparison of developmental trajectories between patients and controls revealed that only processing speed was significantly delayed at 8 years. The rate of development for all variables was lower in SCA patients and age-related decline was accelerated in patients as compared to controls. We speculate that early cerebral haemodynamic compromise may contribute to long term cognitive decline in patients. We also highlight that male SCA patients are particularly vulnerable to SCA disease severity. Hence, timely intervention to stabilise haemoglobin levels, especially in males, should be considered. Limitations include lack of stratification by SCI and age (adults and children separately) and the possibility that we were underpowered to detect effects in females.

Associations of hemolysis and anemia with cardiopulmonary dysfunction in an adult sickle cell disease cohort

BackgroundHemolysis contributes to the anemia of sickle cell disease (SCD). Hemolysis and anemia are distinct but inter-related pathophysiological processes that underlie end-organ dysfunction in this condition. We hypothesized that real-world medical tests would reveal distinct contributions of hemolysis and anemia to certain cardiopulmonary changes in adults with SCD. MethodsWe assessed laboratory and clinical tests of cardiopulmonary function obtained during routine delivery of care in 442 adult SCD patients. We characterized hemolysis by the first principal component (PC1) of reticulocyte percent, lactate dehydrogenase (LDH), aspartate amino transferase (AST) and total bilirubin- the hemolytic component. The relationships of hemoglobin concentration and hemolysis to organ dysfunction were analyzed by multiple regression and path analysis to identify independent associations. ResultsDegree of hemolysis and degree of anemia both associated independently with elevated values for left atrial diameter (LAD) and left ventricular end-diastolic diameter (LVED), and with lower percent predicted forced expiratory volume in first second (FEV1). Degree of hemolysis, but not anemia, associated independently with low values for oxygen saturation, forced vital capacity (FVC), and total lung capacity (TLC)]. Path analysis reinforced the trend by multiple regression for association of both degree of hemolysis and anemia with elevated TRV but not with lower diastolic blood pressure. DiscussionAnalysis of real-world clinical tests suggest that, although they are inter-related, the degrees of hemolysis and of anemia make independent contributions to cardiopulmonary dysfunction and that treatments that specifically target both aspects of sickle cell disease may be of maximal benefit.

Congenital hypothyroidism in a child with Sickle cell anemia (a Case Report)

Sickle cell disease (SCD) is an autosomal recessive disease common in negros and Arab.1 The main pathological cause is the substitution of valine with glutamic acid in the β-chain of haemoglobin producing abnormal haemoglobin called haemoglobin S.1,2,3 The production of this abnormal haemoglobin is responsible for the sickling of red blood cells in deoxygenated conditions. The symptoms include chronic intravascular hemolysis, vaso-occlusion and painful crisis.1,3 Over time, multiple organ damage can develop.1,3,4 Patients with SCD display a defective activation of the alternate complement pathway, the combination of SCD and Autoimmune disease (AID) is rare, potentially underdiagnosed, and its prevalence is unknown.4However, the frequency of coexisting SCD and AID has not been evaluated and the data mainly demonstrated from case reports. Case presentation In this case, we report a rare combination of Sickle cell anaemia and congenital hypothyroidism in a 4 years old Libyan boy. This patient was referred to our Hematology department due to severe anaemia, the patient was diagnosed with Sickle cell anaemia and congenital Hypothyroidismwhen he was 32 days old. Genetic counselling for the family showed sickle cell anaemia, congenital hypothyroidism and celiac disease in the other family members. However, the association of SCD and congenital hypothyroidism were not reported before, this combination could be due to a new gene mutation since the high consanguinity and multiple affection in siblings with SCD, Celiac disease and Hypothyroidism separately or combined. Conclusion SCD and congenital hypothyroidism in toddlers are newly reported in this study. The combination of AID and SCD was detected in many members of this family. Genetic counselling and testing are important in developing countries to educate individuals on SCD and also offer those at risk. Further studies are needed to determine the frequency of congenital hypothyroidism in patients with SCD to look for a new cause of combination.

Inhibition of Hypoxia Inducible Factor Prolyl Hydroxylase By FG-4592 (Roxadustat) Increases Erythropoietic Stimulation in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a devastating monogenic disorder caused by a mutation in the β-globin gene, leading to production of mutant hemoglobin S (HbS). Abnormal polymerization of deoxygenated HbS causes sickling of red blood cells (RBCs), resulting in painful vaso-occlusive crises and chronic hemolytic anemia. Etiologies of chronic anemia in SCD are multifactorial, including intravascular and extravascular hemolysis, inadequate erythropoietin (EPO) production, and abnormal iron homeostasis (Xu and Thein, Blood 2022). Hydroxyurea (HU) is the standard-of-care drug therapy for SCD and primarily reduces HbS polymerization through induction of fetal Hb (HbF) expression. However, it only modestly improves anemia and does not address other causes of anemia in SCD. Additionally, lower Hb levels in SCD are associated with chronic kidney disease (CKD), stroke, and pulmonary hypertension, arguing for the need for additional therapies targeting anemia in SCD. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of drugs developed for treatment of anemia in CKD and end-stage renal disease. HIF-PHIs stimulate erythropoiesis by inhibiting prolyl hydroxylase and von Hippel-Lindau (VHL)-mediated degradation of HIF-α, leading to increased hypoxic signaling and promotion of HIF-induced EPO expression in the kidneys and liver. Stimulation of erythropoiesis also increases production of erythroferrone, the main regulator of hepcidin, and suppression of hepcidin levels by HIF-PHIs may improve iron mobilization. Furthermore, HIF-PHIs can induce HbF expression in erythroblasts derived from CD34+ hematopoietic stem and progenitor cells (HSPCs) (Hsieh et al.,Blood 2007, Feng et al., ASH abstract 2021), suggesting multiple potential therapeutic benefits in SCD. Therefore, we characterized the effect of the HIF-PHI FG-4592 (roxadustat), on HbF induction in CD34+ HSPCs and on hematologic markers, EPO and hepcidin expression in a mouse model of SCD. CD34+ HSPCs from 3 donors were differentiated in vitro to erythroblasts with vehicle, 20 μM of FG-4592, or 50 μM of HU. Flow cytometric analysis on Day 9 showed that FG-4592 treatment increased % F-cells by 1.47-fold (±0.45), compared to a 2.21-fold (±0.89) increase with HU (Fig. 1). We then transplanted lethally irradiated (1000 rads) male 6-8-week-old C57BL/6J mice with bone marrow from transgenic sickle cell mice (Hb SS; Townes model, Jackson Laboratory). Twelve weeks after transplantation, mice with >80% engraftment of the donor HbSS phenotype, determined by Hb electrophoresis, were used for subsequent experiments. Vehicle, 25 mg/kg FG-4592, or 50 mg/kg FG-4592 was administered three times weekly through intraperitoneal injection for 8 weeks. All mice underwent retro-orbital bleeding at baseline (48 h pretreatment), 2, 4, 6, and 8 weeks of treatment for hematological evaluation, including Hb, hematocrit, and reticulocyte % (Fig. 1). At the end of 8 weeks, livers, kidneys, and plasma were collected. Hb and hematocrit levels increased in the 50 mg/kg FG-4592 group compared to the other groups at 4 weeks but decreased by 8 weeks for all groups. Reticulocyte % also decreased in all groups at the 4- and 8-week timepoints. Red cell distribution width (RDW) and platelet count up-trended while mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) down-trended over the 8 weeks, suggestive of an evolving iron deficiency contributing to worsening anemia. Quantitative PCR detected a 182% increase in liver EPO transcript and a 68% increase in kidney EPO transcript in the 50 mg/kg FG-4592 treated group, compared to vehicle. No changes in liver hepcidin transcription were observed in any group. ELISA analysis detected increased plasma EPO and decreased plasma hepcidin levels with increasing doses of FG-4592. None of these trends, except increases in RDW, were statistically significant. Our study is the first to characterize the effect of HIF-PHI drugs in a mouse model of SCD. Preliminary data confirm an effect of FG-4592 on HbF induction and suggest that FG-4592 may increase EPO expression and decrease hepcidin levels in SCD mice. These results highlight the potential of prolyl hydroxylase inhibition as a novel therapeutic approach for chronic anemia in SCD. Future directions include testing higher doses of FG-4592 to induce a stronger erythropoietic effect and monitoring additional markers of iron homeostasis in our mouse model of SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of Cyb5R3 Function Silences Fetal Hemoglobin and Hematocrit Responses to Hydroxyurea in Patients with Sickle Cell Anemia

BACKGROUND: Hydroxyurea (HU) is the first FDA-approved and most widely prescribed drug for sickle cell (SS) patients due to its beneficial effects (decreased Hb polymerization, pain crises and hospitalization) and relative safety profile. While the clinical benefits of HU have been found to associate with increased nitric oxide (NO) and fetal hemoglobin (HbF) in most SS patients, there has been considerable interpatient variability in HbF response to the drug. From this standpoint, HU is imperfectly understood mechanistically. Cytochrome b5 reductase 3 (Cyb5R3), also known as methemoglobin reductase, is an enzyme known to modulate redox signaling in erythrocytes and vascular cells by catalyzing one electron transfer reactions using reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. Functionally, membrane-associated CYB5R3 exerts antioxidant properties by modulating heme iron reduction, plasma membrane coenzyme Q reduction, elongation and unsaturation of fatty acids, cholesterol biosynthesis and drug metabolism, while the soluble form of CYB5R3 reduces red blood cell methemoglobin. Unremitting oxidation of the soluble guanylate cyclase (sGC) heme iron in the absence of sufficient Cyb5R3 activity in vascular smooth muscle has been shown to explain some NO resistance that characterizes pulmonary arterial hypertension and other vasculopathy associated with SCD. More than 40 variants of Cyb5R3 have been identified. Of particular interest is a Cyb5R3 variant (T117S) that contains a threonine to serine substitution in the flavoprotein, which lessens the reductase's activity by roughly 50% compared to wild-type. This variant occurs with high frequency (0.23 minor allele) in persons of African ancestry but is rare (<1%) in all other races. Nothing is known about the role of Cyb5R3 in the anemia of sickle cell disease. We hypothesized that Cyb5R3 may act as a modifier of HbF induction by HU in sickle cell anemia. METHODS: Male and female SS patients (n=633, 12-70 years of age) from the Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (walk-PHaSST) trial were recruited for genetic analysis (University of Pittsburgh IRB Protocol Study 19060255). Genotyping of rs1800457 (T117S SNP) was performed using Taqman genotyping assay. One-way ANOVA and Holms Sidak were used for multiple comparisons of HbF levels by Cyb5R3 genotype and HU treatment status. Intrapatient correlation analyses of HbF and hematocrit (HCT) were conducted using Pearsons r. RESULTS: Comparisons of HbF levels by Cyb5R3 genotype (CC, wild-type; CG, heterozygous wild-type and T117S; GG, homozygous T117S) and HU treatment status (treated vs. untreated) at time of study enrollment (baseline) show that HU is a less potent HbF inducer in the GG group (4.32 mean diff, P=0.344) versus the CC and CG patient groups (CC: 6.027 mean diff, P<0.0001 and CG: 6.914 mean diff, P<0.0001). Correlation analyses showed that as HbF levels increased among HU-untreated patients, the trend was for HCT to decrease. This was true for all SS patients, irrespective of Cyb5R3 genotype (CC: r = -0.336, P<0.0001, n=320; CG: r = -0.437 P<0.0001, n=120; GG: r = -0.253, P=0.136, n=36). However, with HU therapy, the inverse relationship between HbF and HCT was less negative in SS patients carrying at least one copy of the wild-type Cyb5R3 gene (CC or CG). In these individuals, increasing levels of HbF (up to 10%) correlated with positive gains in HCT at HbF greater than 5-6% (CC: r = -0.161, P=0.097, n=107) and 6-7% (CG: r = -0.186, P=0.25, n=40). In patients homozygous for T117S, HCT continued trending downward as HbF increased (GG: r = -0.419, P=0.228, n=10). These data indicate that the positive relationship between HbF and HCT is subject to uncoupling by loss of Cyb5R3 function. In vitro studies with human erythroleukemic K562 cells reveal loss of gamma globin induction by HU treatment when wild-type Cyb5R3 is knocked down by siRNA, indicating a requirement for Cyb5R3 in HU-stimulated HbF induction. SUMMARY andCONCLUSION: Loss of Cyb5R3 activity negatively impacts HbF and HCT in HU treated SS patients. We conclude that the T117S variant may be a useful marker for predicting which SS patients will incur beneficial HbF and HCT responses to HU, thereby improving clinical decision-making about best drug choices for preventing HbS polymerization and treating anemia in SS patients.

Evaluating Equations for Estimated Glomerular Filtration Rate (eGFR) in Patients with Sickle Cell Disease (SCD)

Background: Assessment of kidney function in sickle cell disease poses a unique dilemma, both due to the presence of hyperfiltration and the increased tubular secretion of creatinine, the metabolite most often used in eGFR assessment. In the general population, formulae utilizing serum creatinine have been developed to estimate GFR. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) have developed the most recent formulae. Until recently, the CKD-EPI-2009 creatinine-based formula has been the standard for estimating eGFR and included black race as a variable in the formula. This formula has been noted to overestimate eGFR in SCD patients, but its bias is improved when the black race variable is excluded. Among available formulae, the CKD-EPI-2012 cystatin C-based formula was shown to have the least bias in SCD patients compared to radionucleotide measured GFR but loses its accuracy when combined with creatinine. The recently developed CKD-EPI-2021 formula which excludes race as a variable is becoming the standard formula in most clinical laboratories. In this multicenter study, we sought to evaluate the agreement of various CKD-EPI formulae in SCD patents. Methods: Patients with HbSS or HbSb0 thalassemia between ages 18 and 65 years were enrolled in a prospective cohort study to assess the natural history of kidney disease in SCD. We excluded patients with bone marrow transplantation, diabetic nephropathy, cancer, connective tissue disease, other glomerular diseases, known infection with hepatitis (B or C) or HIV, and end-stage kidney disease on dialysis. Participants were enrolled during routine clinical visits at four centers. At baseline, blood samples and urine were obtained, and kidney function was assessed using each sites clinical laboratory to measure serum creatinine. Serum cystatin C was also measured at baseline using a commercially available ELISA kit. We calculated eGFR using each of the CKD-EPI formula, excluding the race variable in the CKD-EPI-2009 formula. Spearman's rank correlation coefficients were calculated for each formula compared to the others. Prevalence of CKD was assessed using the CKD-EPI-2009, CKD-EPI-2012 (cystatin C) and CKD-EPI-2021 formulae and defined as the presence of albuminuria or an eGFR ≤ 90 ml/min/1.73m2. Prevalence of CKD derived from the CKD-EPI-2012 was compared to the other two formulae using McNemar's Chi-squared test with 95% confidence intervals determined using bootstrap replications. Results: Our cohort enrolled 284 subjects with HbSS or HbSb0 thalassemia. For these analyses, 277 patients with baseline serum creatinine were included. The mean age of included subjects was 32.9 (SD ±11.5) years and 57% were female (N=158). The median eGFR of these subjects using the creatinine-based CKD-EPI-2009 formula was 123.9 (IQR 105.9, 134.0) ml/min/1.73m2. Cystatin C was available in 270 patients and had median value of 0.72 (IQR 0.59, 0.92) mg/L. Correlation between the several eGFR formulae are presented in Table 1. CKD-EPI-2009 and CKD-EPI-2021 creatinine-based formulae were very highly correlated with each other; both had similar but less correlation with the CKD-EPI-2012 cystatin C-based formula, which has been shown previously to have the least bias when compared to measured GFR. The prevalence of CKD using the creatinine-based CKD-EPI-2009 formula was 51.3% and 50.5% using the creatinine-based CKD-EPI-2021 formula. When assessed with the cystatin C-based CKD-EPI-2012 formula, CKD prevalence was higher at 55.7% (p<0.0001) than that seen with both of creatinine-based CKD-EPI formulae. Conclusions: When comparing the new CKD-EPI-2021 creatinine-based formula to the CKD-EPI-2009 formula (excluding the black race variable), eGFR calculation remains very similar in SCD patients. The CKD-EPI-2009 formula is well recognized to overestimate eGFR in SCD patients. Our data would suggest the newer formula would have similar bias. Using the cystatin C-based CKD-EPI-2012 formula, the prevalence of CKD is higher in this SCD cohort than using creatinine-based CKD-EPI formulae, although our data are limited by lack of directly measured GFR. We anticipate the newer CKD-EPI-2021 creatinine-based formula would similarly underestimate prevalence of CKD in SCD. Use of cystatin C for assessing kidney function in SCD patients should be considered, and resources should be utilized to make this measure more widely and readily available. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Uremic Metabolites Are Associated with Albuminuria and Estimated Glomerular Filtration Rate (eGFR) in Sickle Cell Disease

Introduction: Chronic kidney disease (CKD) is common in patients with sickle cell disease (SCD) and is associated with increased morbidity and mortality. As albuminuria and changes in estimated glomerular filtration rate (eGFR) occur well after substantial functional and structural damage have occurred, identification of novel biomarkers which could predict the development of CKD is necessary. Previous studies have shown an increase in the level of the uremic metabolite indoxyl-sulfate (IS) in urine in patients with SCD. IS and other uremic metabolites are cleared by active secretion in kidney tubule cells and their accumulation in these cells leads to increased oxidative stress, and apoptotic and necrotic cell death. In this single center, cross-sectional study, we investigated the associations between both urinary levels of uremic toxins and biomarkers of kidney tubular injury with persistent albuminuria and eGFR in patients with SCD. Methods: The participants represent a subset of patients with HbSS or HbSβ0 thalassemia enrolled in a prospective cohort study to assess the natural history of kidney disease in SCD. We excluded patients with diabetic nephropathy, cancer, connective tissue disease, other glomerular diseases, known infection with hepatitis (B or C) or HIV, end-stage kidney disease on dialysis and who had undergone bone marrow transplantation. Participants were enrolled during routine clinic visits. Urine levels of uremic toxins, indoxyl sulfate (IS) and trimethylamine-n-oxide (TMAO), and tubular injury biomarkers, monocyte chemoattractant protein-1 (MCP-1; also called CC-chemokine ligand 2 [CCL2]) and neutrophil gelatinase-associated lipocalin (NGAL) were measured using quantitative ELISA assay. All values obtained for urine biomarkers were normalized to urine creatinine. Persistent albuminuria was defined based on at least 2 of 3 spot urine albumin/creatinine ratio (ACR) values ≥30 mg/g of creatinine or a single value ≥100 mg/g. Glomerular filtration rate was estimated using the 2009 creatinine-based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. As the distribution of the biomarkers were skewed, the data were log-transformed. Associations between both ACR and eGFR with each of the urine biomarkers were assessed using linear regression. Results: One hundred and sixteen subjects, 26 (22.4%; ACR - median 276.5 mg/g [IQR: 156, 802]) with persistent albuminuria (PA), and 90 (77.6%; ACR - median 13 mg/g [IQR: 6.5, 25.5]) without persistent albuminuria (non-PA) were evaluated. Subjects with PA appeared to be older and had lower hemoglobin and fetal hemoglobin levels, but higher reticulocyte counts, lactate dehydrogenase levels and systolic and diastolic blood pressures than subjects with non-PA (Table 1). Log-transformed urine levels of IS (β=0.52, 95% confidence interval [CI]: 0.08, 0.96, p=0.02), MCP-1 (β=0.65, 95% CI: 0.29, 1.01, p=0.0006) and NGAL (β=0.25, 95% CI: 0.02, 0.49, p=0.03) were positively associated with log-transformed ACR (Table 2). Log-transformed urine levels of IS (β=-8.0, 95% CI: -14.7, -1.3, p=0.02), MCP-1 (β=-11.6, 95% CI: -16.9, -6.3, p<0.0001) and NGAL (β=-6.0, 95% CI: -9.4, -2.6, p=0.0008) were inversely associated with eGFR. Log-transformed urine level of TMAO was not associated with either ACR or eGFR. Conclusion: This study demonstrates a novel association of IS with albuminuria and eGFR in patients with SCD. In addition, we confirm the association of biomarkers of tubular injury, MCP-1 and NGAL, with persistent albuminuria and eGFR in SCD. Future studies will determine whether uremic metabolites may predict the development of albuminuria in patients with SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Compact Point of Care Ultrasound Reveals Hemodynamic Abnormalities in Children with Sickle Cell Disease

In sickle cell disease (SCD), kidney related morbidity and mortality are significant, with ~80% of patients displaying signs of chronic kidney disease (CKD) by age 401 and kidney related mortality being the third leading cause of death (16.4%)2. Children with SCD are also prone to kidney disease with 20% of 12 years-old displaying microalbuminuria4 and enuresis and hyposthenuria commonly observed in infants4. Current clinical guidelines are insufficient as they ignore pediatric disease until the development of late signs like proteinuria or large drops in eGFR have occurred5. A major limitation in the assessment of kidney disease in children with SCD is the incomplete understanding of disease progression and the lack of suitable biomarkers to discriminate health and disease. Serum creatinine based eGFR is the most common biomarker of kidney function but is poorly validated and relatively insensitive in SCD patients due low muscle mass and hyperfiltration6. Point of care ultrasound (POCUS) offers a non-invasive alternative to blood based biomarkers. POCUS has proven clinical utility in decreasing primary and secondary stroke7. Additionally, POCUS technology now offers compact, cartless systems, with tablet and smartphone compatibility, multiprobe functionality in a single probe and an economical price point (<3000$)8. Therefore, the goals of this study were two-fold. 1) Determine the feasibility of renal assessment using compact POCUS in children with SCD in a clinical setting. 2) Establish normative values for possible compact POCUS biomarkers that could supplement existing biomarkers of kidney disease in SCD. Methods: Studies were performed with informed consent under an IRB approved protocol. 18 patients with SCD were recruited during their routine clinical visit to undergo a 10-20 minute ultrasound examination. All POCUS exams were performed in supine position, on the right kidney by an experienced imaging researcher (AB, +10 years). Images were acquired with a Butterfly IQ+ (Burlington, MA, USA) system and an Apple Smartphone (Cupertino, CA, USA). The abdominal preset and B-mode imaging was used to determine the long axis renal length and echogenicity of the renal/liver parenchyma. The cardiac preset, pulsed Doppler mode was used to determine the peak systolic velocity (PSV), end diastolic velocity (EDV) and resistive index (RI = (PSV-EDV)/PSV) of the main right renal artery. Images were saved to a cloud based, HIPPA protected database, downloaded to a local workstation and processed using custom MATLAB scripts. An angle correction of 1/cos(60°) was applied to the uncorrected velocity values. Results: Renal length and serum creatinine was measured in 18 patients with SCD (3 HbSC, 15 HbSS) and pulsed Doppler estimates were obtained in 14 of 18 subjects (hemoglobin 9.9±2.3,12 male). The average renal length was 8.7±1.3 cms and serum creatinine was 0.69±0.16 and showed a statistically significant increase with respect to age (Figure 1). The average angle corrected main renal artery PSV was 74.5±64.4 cm/s and RI was 0.49±0.33 and both decreased with respect to age (Figure 1). The average echogenicity ratio between kidney and liver parenchyma was 0.64±0.14 and was uncorrelated with age. Discussion: This work demonstrates that high quality structural and hemodynamic images can be acquired with low cost, compact POCUS in a clinical setting. The age dependent increase in serum creatinine9 and kidney length10 were in good agreement with prior cart-based sonography work in healthy children. Conversely, the age dependent drop in main renal artery PSV and RI was in opposition to work in healthy children that showed no change11 or an increase with age12. The clinical significance of abnormally elevated velocity in young children with SCD and its impact on the development of sickle nephropathy should be studied in more detail. Future work comparing cross-sectional and longitudinal variability in compact POCUS and physiologic biomarkers of kidney function in both pediatric and adult subjects with and without SCD is ongoing.

Association of Novel Urinary Protein Biomarkers with Persistent Albuminuria in Patients with Sickle Cell Disease

Introduction: Standard assessments for kidney disease, albuminuria and decline in estimated glomerular filtration rate (eGFR), occur well after substantial functional and structural damage have ensued. Chronic kidney disease (CKD) is common in patients with sickle cell disease (SCD). With its association with increased morbidity and mortality, identification of novel biomarkers that could predict early development of CKD offers opportunities to modify risk factors in hopes of attenuating kidney function loss and decreasing mortality risk. Methods: The participants of this exploratory study represent a subset of patients with HbSS or HbSβ0 thalassemia enrolled in a prospective cohort study to assess the natural history of kidney disease in SCD. We excluded patients with diabetic nephropathy, cancer, connective tissue disease, other glomerular diseases, known infection with hepatitis (B or C) or HIV, end-stage kidney disease on dialysis and those who had undergone bone marrow transplantation. Participants were enrolled during routine clinic visits. Urine levels of clusterin [CLU; also referred to as apolipoprotein J], retinol-binding protein 4 [RBP4], alpha-1-microglobulin [A1M], interleukin-18 [IL-18], beta-2 microglobulin [B1M], insulin-like growth factor-binding protein 7 [IGFBP7], tissue inhibitor of metalloproteinases-2 [TIMP2], angiotensinogen [AGT], podocalyxin, and cysteine-rich with EGF-like domains 2 [CRELD2] were measured using quantitative ELISA assay. All values of urine biomarkers were normalized to urine creatinine. Persistent albuminuria (PA) was defined based on presence of at least 2 of 3 spot urine albumin/creatinine ratio (ACR) values ≥30 mg/g of creatinine or a single value ≥100 mg/g. Glomerular filtration rate was estimated using the 2009 creatinine-based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. Medians are presented with the interquartile range [IQR]. Associations between ACR and eGFR with each urine biomarker were assessed using linear regression. Levels of urinary biomarkers were compared using the Wilcoxon-Mann-Whitney test. Results: Thirty-seven subjects, 13 (35.2%) with PA (ACR - 147mg/g [IQR: 136.4, 223]), and 24 (64.9%) without PA (non-PA) (ACR - 12 mg/g [IQR: [6, 20]) were evaluated. Subjects with PA were older, had higher blood urea nitrogen (BUN), blood aspartate transaminase, and lower eGFR (Table 1). Urinary levels of CLU, RBP4, A1M, and AGT normalized to urine creatinine were significantly higher in subjects with PA vs. those with non-PA (Table 2). In univariate analysis, significant associations were identified between A1M and ACR (p=0.035) as well as between AGT and ACR (p<0.0001) (Table 2). In multivariable analyses, only AGT remained significantly associated with ACR (p=0.0003). Conclusion: The higher urinary levels of RBP4 and A1M, both low molecular weight proteins freely filtered in the glomerulus and reabsorbed by kidney proximal tubule cells, in subjects with PA suggests the presence of tubular injury. Higher levels of clusterin, a ubiquitously expressed glycoprotein that is rapidly upregulated upon acute kidney damage and present in de-differentiated kidney tubule cells, also indicates tubular injury in subjects with PA. Urinary AGT, the only precursor of all angiotensin peptides, is independently associated with ACR in this cohort. Higher levels of AGT indicates increased intrarenal renin-angiotensin system activity in subjects with PA. More studies are required to evaluate the association of these biomarkers with persistent albuminuria in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Managing Kidney Disease in Patients with Sickle Cell Disease: A Quality Initiative at the Cleveland Clinic

Background Sickle cell disease (SCD) when complicated by sickle cell nephropathy and chronic kidney disease (CKD) is associated with high morbidity and mortality. Early intervention with appropriate specialist care and timely evaluation for transplant are essential to improve outcomes. There is considerable variability in the screening, diagnosis and management of renal complications in SCD. Current guidelines from the NHLBI (2014), ASH (2019) and expert opinion endorse (1) annual screening for proteinuria, (2) the initiation of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II receptor blockers (ARB) in patients with significant proteinuria, (3) early involvement of nephrology for declining renal function, (4) testing for alternative etiologies of kidney disease, (5) referral for renal transplant in advanced CKD with regular red cell exchange in those being considered for transplant, and (6) combination therapy with hydroxyurea and erythropoiesis-stimulating agents (ESA) for patients with worsening anemia from CKD. We sought to audit our care of SCD patients along these metrics to devise effective interventions to improve outcomes in SCD patients with comorbid kidney disease. Methods We conducted a retrospective quality review of renal disease screening, referral, and management in adults with SCD who were seen in the outpatient setting at the Cleveland Clinic in the past 3 years. Results We reviewed 258 adults with SCD who were seen between July 2019 and July 2022. Of these, 16 patients had insufficient laboratory data and were excluded from further analysis. Our final cohort of 242 patients was comprised of 169 (70%) female patients and the median age was 42 (18-90) years. The majority [213 (88%)] had appropriate screening for proteinuria. Of 46 patients with significant proteinuria, 18 (39%) were on ACEi/ARB in part reflecting challenges with tolerance (hyperkalemia, cough, hypotension). CKD was present in 43 (18%) patients; 25 (58%) had CKD stage 3a, 5 (12%) stage 3b, 6 (14%) stage 4, and 7 (16%) were on dialysis. Nephrology was involved in 27 (63%) of those with CKD. Most [31 (72%)] were evaluated for other causes of CKD, however the extent of testing was inconsistent. Referral for renal transplant evaluation was made in 7 (16%) patients with CKD and approximately half [3 (43%)] of these patients were receiving regular red cell exchange transfusions. Only 2 (5%) patients received a kidney transplant. Of the subset of 13 patients with advanced CKD stages 4 or 5, 9 (69%) were receiving ESA and 3 (23%) were on combination hydroxyurea and ESA. In our larger cohort of 242, an additional 30 (12%) patients had isolated persistent microscopic hematuria and 44 (18%) patients demonstrated a trend of chronically rising creatinine within the laboratory normal range. Additional analysis is planned to evaluate screening and management in this at-risk cohort with more subtle presentation. Discussion The management of renal disease in SCD is complex and a paucity of data to support evidence-based guidelines presents a major challenge. We found that rates of screening for proteinuria and declining renal function in SCD patients were high, however the subsequent management was inconsistent. CKD in SCD is highly morbid, and patients with SCD continue to experience low rates of transplant evaluation and eligibility with devastating consequences. A root cause analysis, patient and provider education campaigns, and additional activities are planned to improve the management of kidney disease in patients with SCD.

Altered Cellular Sedimentation during Apheresis Impacts Outcomes of Peripheral Blood Stem Cell Collection Efficiency in Patients with Sickle Cell Disease

Recent reports have demonstrated the potential for gene therapy as a therapeutic option to achieve a durable cure in patients with sickle cell disease (SCD). However, for these emerging cell therapies, relatively large numbers of peripheral blood hematopoietic stem cells (PB HSCs) are required to generate a therapeutic dose. Outcomes of stem cell collections in patients with SCD are highly variable, at times yielding far fewer numbers of HSCs in the product than predicted by the number of PB CD34+ cells. While the CXCR4 antagonist plerixafor has emerged as a safe and reliable drug to mobilize CD34+ cells in patients with SCD, collection failures continue to occur requiring additional cycles or failure to generate a gene therapy product. We sought to understand the mechanics affecting stem cell sedimentation in real time and posited that SCD disease associated changes to red cells results in altered HSC sedimentation during apheresis, negatively impacting collection. Methods Autologous PB stem cell collections were performed in patients with SCD following mobilization with 240ug/kg plerixafor. To uncover the mechanisms impacting apheresis efficiency for collecting PB HSCs, we devised a method to interrogate the sedimentation of cells in peripheral blood in real time, by interrupting the return collection circuit line. Cellular composition analyses of the collecting interfaces were performed including CD34+ enumeration by flow cytometry, CBC and differential, reticulocyte counts, and surface CD47 expression of red blood cells (RBCs). Results Patients were mobilized with 240ug/kg of plerixafor up to 6 hours before apheresis collection. We collected fewer cells than predicted (3.78 million cells/kg) in these patients with an overall average apheresis collection efficiency of 38%, and a strikingly wide range of 2%-107%. We determined that the lower collection efficiency outcomes were primarily derived when using standard apheresis interface settings. The pre-apheresis PB CD34+ counts were less predictive of total CD34+ cells collected (p=0.012) in patients with SCD (r2=0.2557, p=0.0023) when compared to historical control patients without SCD (r2=0.7638, p<0.0001). Real time measurement of cell counts at standard (Hematocrit 3%, S) and deep (Hematocrit 5%, D) collecting interfaces of the apheresis machine showed a significant decrease in lymphocytes (p=0.0146), increase in monocytes (p=0.0496), and increase enrichment in CD34+ cells at the D interface (Panel A). Enrichment of CD34+ cells correlated with monocyte enrichment and (n=21, r2=0.6706, p<0.0001) and an increased fold change in CD34+ cells between the S and D interfaces correlated with higher PB reticulocyte percentage (r2=0.5295, p=0.0111). Further analysis of the individual collection interfaces revealed that most RBCs were HbS containing despite a PB HbS concentration <30%, consistent with an enrichment for shorter lived sickle red cells. CD47 expression has been previously reported in higher concentrations on the surface of younger RBCs during erythropoiesis. Surface expression of CD47 was found to be significantly higher at both the S and D interfaces compared to the PB of patients, indicating that the accumulation of a younger population of RBCs at the collecting interfaces. As microcytic RBCs have been associated with poor collection efficiencies, the SCD red cell hemoglobin concentration in the PB was used as a marker for size and a significant correlation was determined (r2=0.6086, p=0.0001). Patients with SCD can mobilize PB HSC in response to plerixafor but demonstrate specific characteristics that impede the collection of stem cells. During apheresis, CD34+ stem cells sediment at a deeper hematocrit interface in the buffy coat as a result of displacement by the younger patient-derived population of RBCs. The relative shift in erythrocyte sedimentation requires a corresponding alteration in collection strategies to improve the collection efficiency. In addition to red cell age, other factors that affect red cell buoyancy including iron status or additional globin gene mutations negatively impact apheresis stem cell collections. Full assessment of red cell parameters can identify patients that are at risk for poor stem cell collection outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of MRP1 Induces Fetal Hemoglobin through NRF2 Activation to Protect Human Erythroid Cells from Sickling

Introduction: Sickle cell disease (SCD) is a genetic disorder caused by a single nucleotide substitution (Glu6→Val) in the HBB gene coding for the β-globin of the hemoglobin A (HbA; α2β2) in erythroid cells. This point mutation leads to abnormal hemoglobin (HbS), which polymerizes in low oxygen condition and, ultimately, causes sickling of circulating erythrocytes leading to hemolysis, vascular inflammation, and painful vaso-occlusive crises. Hereditary persistence of fetal hemoglobin (HbF; α2γ2) in sickle cell patients significantly reduces clinical symptoms by preventing polymerization of HbS and subsequent cell sickling. Therefore, raising HbF expression in SCD patients is a promising therapeutic strategy. However, hydroxyurea, the standard of care for SCD and only FDA-approved HbF inducer, carries concerns for carcinogenicity-associated effects and the non-responders rate for this drug is relatively high (~30%). Consequently, there is a large unmet need for new therapies in SCD. Methods: To discover potential new HbF inducers, we conducted a genome-wide association study of HbF levels in 11,004 healthy donors from the INTERVAL study. We identified ABCC1, which encodes the multidrug resistant protein 1 (MRP1) and efflux pump for glutathione, as a putative causal gene modulating HbF expression. We detected a specific missense variant in ABCC1 which increases HbF expression in erythrocytes and strongly associates with plasma glutathione levels (β=0.8SD, P=3x10-38). To elucidate the role of MRP1 in HbF regulation, we performed a CRISPR-Cas9-mediated ABCC1 knockdown in erythroid HUDEP-2 cells and CD34+ hematopoietic stem cells and quantified the expression of HbF by flow cytometry, Western blot and high-performance liquid chromatography (HPLC) after erythroid differentiation (2-phase culture). We also generated HUDEP-2 clones homozygous for the p.R433S variant using CRISPR-Cas9 gene editing and measured HbF levels using the methods previously described. In CD34+ cells, MRP1 effect on HbF expression was also evaluated by using the selective MRP1-inhibitor, MK571. Transcriptomic, proteomics and phospho-proteomic analysis were performed to identify the mechanism of action involved in HbF induction after MRP1 inhibition. Expression of erythroid differentiation markers GATA1, ALAS2, LRF and Band 3 was assessed by Western blot to evaluate the impact of MRP1 inhibition on erythroid differentiation. Finally, we conducted sickling assays on MK571-treated CD34+ cells from SCD patients. Results:ABCC1 knockdown (90% gene silencing compared to control) in HUDEP-2 cells increased intracellular glutathione level (7−fold, P<0.01) and HbF (2.5−fold, P<0.05), as shown by flow cytometry, HPLC and Western blot analysis, after 7-day differentiation. Similarly, HUDEP-2 clones homozygous for the ABCC1-R433S allele presented an increase in intracellular glutathione (6-fold, P<0.05) and HbF expression levels (6-fold, P<0.01) compared to non-treated cells. In CD34+ cells knockdown for ABCC1 (95% gene silencing compared to control), HbF was increased (1.5−fold, P<0.05) after 14-day differentiation. Furthermore, pharmacological inhibition of MRP1, by MK571, in CD34+ cells led to an induction in glutathione (5-fold, P<0.01) and HbF (2-fold, P<0.05), and prevented sickling of CD34+ cells from SCD patients (2-fold decrease, P<0.05) in hypoxic condition (2% O2), without impacting erythroid differentiation. Finally, transcriptomic, proteomic, and phospho-proteomic integrated analyses in MK571-treated CD34+ cells revealed an activation of the NRF2-mediated oxidative stress pathway. Addition of ML385 (10µM), an NRF2 inhibitor, in MK571-treated CD34+ cells completely abolished HbF induction, confirming a NRF2-dependent HbF induction. Conclusion: In summary, we identified MRP1 as a HbF modulator in human erythroid cells and demonstrated that MRP1 inhibition induces HbF and prevents sickling of CD34+ cells through NRF2-mediated oxidative stress pathway activation without affecting erythroid differentiation, establishing MRP1 as a potential new therapeutic target for SCD. Future directions include replication of the ABCC1 genetic association in an independent cohort, and in vivo studies aiming to confirm the efficacy of MRP1 inhibition on improving SCD disease biomarkers.

ASSOCIATION BETWEEN HEMOGLOBIN LEVELS AND END-ORGAN DAMAGE IN SICKLE CELL DISEASE: A RETROSPECTIVE LINKED PRIMARY AND SECONDARY CARE DATABASE ANALYSIS IN ENGLAND

Sickle cell disease (SCD) is a chronic, multifaceted blood disorder that affects the oxygen-carrying protein hemoglobin (Hb). Along with chronic hemolysis and vaso-occlusion that often accompanies SCD, low Hb levels may contribute to downstream end-organ damage (EOD). Emerging evidence suggests an association between chronic hemolytic anemia in SCD and both short-term and long-term adverse outcomes, including EOD. Therefore, treatments that directly increase Hb may improve patients’ acute symptoms while reducing the risk for long-term disease-related effects known to contribute to morbidity and mortality. This study aims to ascertain the association between Hb level variation and EOD and clinical conditions typically associated with SCD (stroke, pulmonary hypertension, chronic kidney disease [CKD], end-stage renal disease [ESRD], leg ulcers, and pneumonia) in a real-world data set. The Clinical Practice Research Datalink (CPRD) and the Hospital Episode Statistics (HES) databases in England were used to retrospectively identify linked Hb data among patients with SCD (1 April 2007 to 31 March 2019). Recurring results in any 90-day period were averaged. Logistic regression adjusted with demographic and clinical variables was used to determine odds ratios (ORs) of developing each EOD outcome or clinical complication. ORs, p values, 95% confidence intervals (CIs), and areas under the receiver operating curve (AUC-ROCs) and corresponding 95% CIs were reported. Of 11,936 patients identified, 5379 (45%) had Hb results recorded in the CPRD. The logistic model for risk assessment of all outcomes studied indicated that an increase in Hb among patients with SCD was associated with a statistically significant (p < 0.001) decrease in adjusted ORs per 1 g/dL increase in Hb. Except for ESRD, leg ulcers, and pneumonia, the adjusted models showed statistically significant increases in AUC-ROC compared with the unadjusted models, signifying that the adjusted models were more robust and further verified a statistical OR after adjustment. Increased Hb was associated with decreased risk of respiratory-related events. Pulmonary hypertension had the strongest effect with the most robust model (OR adjusted of 0.66). For pneumonia, OR adjusted was 0.77. For renal insufficiency, ORs for developing CKD or ESRD were significantly lower than 1 (OR adjusted = 0.73 and OR adjusted = 0.75, respectively). After adjusting for clinical and demographic factors, the model predicted a lower risk for stroke (OR adjusted = 0.89). The estimated AUC-ROCs suggest that the adjusted model is more robust in stroke, hypertension, and CKD. These findings did not change when testing for other covariates such as deep vein thrombosis. Among patients with SCD, an increase in Hb of 1 g/dL was associated with a statistically significant reduction in risk for 6 common EOD outcomes and clinical conditions. The results were more pronounced after adjusting for demographic and clinical covariates (based on clinical opinion and literature review) and further assessed with a bivariate analysis. The results were obtained from a representative real-world evidence dataset analyzed over a 12-year period, which was sufficient to observe EOD events, and are generalizable to the UK and similar populations. Our findings support the use of therapeutics that increase Hb in patients with SCD to protect against deleterious organ damage. This study and its analysis were conducted by CorEvitas LLC and sponsored by Global Blood Therapeutics.

S3161 Sickle Cell Hepatopathy: A Rare Complication of Sickle Cell Anemia

Introduction: Sickle cell hepatopathy (SCH) describes the spectrum of hepatobiliary complications seen in sickle cell disease (SS). The etiology is multifaceted; repeated sickling causes liver vaso-occlusion and sinusoidal obstruction, which contributes to ischemic hepatic damage. SCH treatment is supportive; it includes supplemental oxygen, intravenous fluids and exchange transfusions. We present the diagnosis and management of a rare case of SCH. Case Description/Methods: Our patient is a 30-year-old male with SS anemia complicated by avascular necrosis of the shoulder, infrequent pain crises and cholecystectomy, who presented to our ED with fevers >100.4F, abdominal pain, acholic stools, dark urine and jaundice. Initial workup showed transaminitis, cholestasis, with an alkaline phosphatase 189, ALT 104, AST 145 and a total bilirubin 46.5. Abdominal ultrasound and CT did not show any acute hepatic causes for these lab abnormalities. Fevers prompted an infectious workup; viral hepatitis markers and blood cultures were negative. Autoimmune processes were ruled out with normal ANA, ASMA and immunoglobulins. He was given supportive care via intravenous fluids. The fevers resolved, but his abdominal pain and jaundice persisted. His t-bili increased without any evidence of infection or renal failure. Hepatology was concerned that he had intrahepatic cholestasis as a result of his SS. They recommended an exchange transfusion and considered a liver transplant. Our patient had many antibodies in his blood, increasing the risk of complications from an exchange transfusion. We deferred a liver biopsy due to the known increased risk of bleeding in SS patients and a lack of impact on management. We continued supportive care, and his liver function improved. Discussion: Acute sickle cell hepatopathy can range from an intra-hepatic sickle cell crisis, acute liver sequestration, and intrahepatic cholestasis; treatments include supportive care and exchange transfusions. Severe cases may require a liver transplant. We were initially concerned for acute intrahepatic cholestasis for our patient, however, his fevers did not persist and he did not have evidence of multi-organ system failure. There was also no evidence of worsening of sickling on a peripheral smear. If his lab abnormalities and symptoms worsened, we would consider doing exchange transfusions or a liver transplant. He improved with conservative measures, and this is an example of a rare complication of a serious disease.

Amiodarone improves anemia in a murine model of sickle cell disease and is associated with increased erythrocyte bis(monoacylglycerol) phosphate

Sickle cell disease (SCD) is associated with altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 μM) or medroxyprogesterone (6.75 μM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP.

Plasma immune mediators as laboratorial biomarkers for Sickle Cell Disease patients according to the hydroxyurea therapy and disease severity

In the present work, the impact of Sickle Cell Disease (SCD) degrees of severity, as well hydroxyurea treatment on the systemic immunological signatures of patients was evaluated. Based on a high-throughput chemokine, cytokine and growth factor multiplex analysis, it was possible to obtain the systemic immunological profile of patients with SCD (n = 40), treated or not with hydroxyurea, as compared to healthy controls (n = 40). Overall, SCD patients with severe disease displayed increased levels of almost all biomarkers analyzed. Our data demonstrated that CXCL8, CCL3 and CXCL10 were pointed out as universal biomarkers of SCD. The results also indicated that HU-untreated patients with indication of HU-therapy display a more prominent increase on plasma immune mediators in a similar way as those with severe SCD disease. Together, these findings provided a comprehensive landscape of evidence that may have implications for further therapeutic strategies and SCD clinical management.

Development and characterization of a preclinical total marrow irradiation conditioning-based bone marrow transplant model for sickle cell disease

Sickle cell disease (SCD) is a serious global health problem, and currently, the only curative option is hematopoietic stem cell transplant (HCT). However, myeloablative total body irradiation (TBI)-based HCT is associated with high mortality/morbidity in SCD patients. Therefore, reduced-intensity (2–4 Gy) total body radiation (TBI) is currently used as a conditioning regimen resulting in mixed chimerism with the rescue of the SCD disease characteristic features. However, donor chimerism gradually reduces in a few years, resulting in a relapse of the SCD features, and organ toxicities remained the primary concern for long-term survivors. Targeted marrow irradiation (TMI) is a novel technique developed to deliver radiation to the desired target while sparing vital organs and is successfully used for HCT in refractory/relapsed patients with leukemia. However, it is unknown if TMI will be an effective treatment for a hematological disorder like SCD without adverse effects seen on TBI. Therefore, we examined preclinical feasibility to determine the tolerated dose escalation, its impact on donor engraftment, and reduction in organ damage using our recently developed TMI in the humanized homozygous Berkley SCD mouse model (SS). We show that dose-escalated TMI (8:2) (8 Gy to the bone marrow and 2 Gy to the rest of the body) is tolerated with reduced organ pathology compared with TBI (4:4)-treated mice. Furthermore, with increased SCD control (AA) mice (25 million) donor BM cells, TMI (8:2)-treated mice show successful long-term engraftment while engraftment failed in TBI (2:2)-treated mice. We further evaluated the benefit of dose-escalated TMI and donor cell engraftment in alleviating SCD features. The donor engraftment in SCD mice completely rescues SCD disease features including recovery in RBCs, hematocrit, platelets, and reduced reticulocytes. Moreover, two-photon microscopy imaging of skull BM of transplanted SCD mice shows reduced vessel density and leakiness compared to untreated control SCD mice, indicating vascular recovery post-BMT.