Sickle Cell Anemia Disease Research Articles

Cardiac Disease in Egyptian Thalassemia Major and Sickle Cell Disease Adolescents in relation to Vitamin D Status Using Echocardiogram & Ultrafast Magnetic Resonance Imaging

Abstract Background Cardiovascular complications are well recognized in patients with β-thalassemia major (β-TM) and sickle cell disease (SCD). Vitamin D deficiency is prevalent in patients with transfusion-dependent thalassemia and there is an association between vitamin D deficiency and both left ventricular function and iron overload in thalassemia. However, the relationship between vitamin D and cardiac iron is not well studied in Egypt and the role of vitamin D in cardiac disease in SCD and β-TM needs to be assessed. Aim To assess vitamin D status in children and adolescents with SCD and β-TM in relation to cardiac complications using echocardiography and cardiac magnetic resonance (CMR) T2*. Methods Forty-one patients with SCD and 35 patients with β-TM without symptomatic cardiac disease were studied stressing on history of splenectomy and transfusion history. Markers of hemolysis, serum ferritin and vitamin D levels were measured. Liver iron concentration (LIC) was assessed by magnetic resonance imaging (MRI). Screening for cardiovascular abnormalities was performed by the non-invasive Doppler echocardiography and CMR was used to assess myocardial iron overload. Results Echocardiography showed that cardiac functions were significantly impaired in β-TM patients compared with SCD while the frequency of pulmonary hypertension risk (elevated TRV ≥2.5 m/sec) was consistent between both β-TM and SCD groups. Cardiac T2* (which reflects cardiac iron overload) was significantly lower while myocardial iron concentration (MIC) was significantly higher in β -TM patients compared with SCD group. Vitamin D levels were decreased in both SCD and β-TM patients than control levels; however, SCD patients had significantly lower vitamin D levels. Low vitamin D levels were significantly associated withincreased risk of pulmonary hypertension in the SCD group while In β-TM group, vitamin D levels were significantly lower in patients with cardiac disease and those with serum ferritin levels ≥ 2500 µg/L. A significant evidence of diastolic dysfunction was found in SCD and β-TM patients who had low serum vitamin D level < 20 ng/mL. Vitamin D levels were negatively correlated to serum ferritin and LIC while positively correlated to cardiac T2*. Conclusion Low vitamin D levels were associated with cardiopulmonary dysfunction in adolescents with SCD and β-TM. Vitamin D deficiency in those patients was linked to cardiac iron overload leading to iron induced cardiomyopathy. Proper chelation therapy is mandatory to improve cardiac disease, iron overload and consequently, vitamin D status in β-TM patients.

Determinants of the haemoglobin level in patients with sickle cell disease living in sub-Saharan Africa: Major impact of the country of residence and independent effects of leucocyte and platelet counts and haemolysis.

The degree of anaemia in sickle cell disease (SCD) is a well-known contributor to morbidity and mortality. We aimed to explore the factors affecting haemoglobin (Hb) level in African SCD patients, considering haemolysis biomarkers (LDH and bilirubin level, and reticulocyte count), leucocyte and platelet counts and socio-demographic characteristics (gender, age group, country of residence and BMI). The research was part of the CADRE multinational cohort and involved 3699 SCD patients living in Mali, Senegal, Ivory Coast, Democratic Republic of Congo, Gabon and Cameroon: 2936 SS/Sβ0, 587 SC and 176 Sβ + patients with median Hb level of 8, 11.3 and 11.2 g/dL respectively (p < 0.001). In multivariate analysis conducted in 1394 SS/Sβ0 patients, living in Cameroon, female gender, lower BMI, higher haemolysis markers (especially LDH) and higher leucocyte and platelet counts were independently associated with lower Hb level (all p < 0.05). In 497 SC and 156 Sβ + patients, female gender (p < 0.001), lower BMI (p < 0.05) and higher platelet counts (p < 0.001) were independently associated with lower Hb level. Anaemia in African SCD patients is not only associated with haemolysis but also with the country of residence, lower BMI and leucocyte or platelet counts which might reflect inflammation related to infectious burden in the region.

Thrombin activatable fibrinolysis inhibitor plasma levels and TAFI Thr325Ile genetic polymorphism in a cohort of Egyptian sickle cell disease patients and impact on disease severity.

Thrombin is a critical protease modulating thrombosis as well as inflammation, which are one of the main pathophysiological mechanisms in sickle vasculopathy, and its levels were reported to be high in sickle cell disease (SCD). The thrombin-thrombomodulin complex activates the TAFI inhibitor of fibrinolysis, which acts by reducing plasmin affinity for its substrate thus hindering fibrinolysis. We aimed to determine the influence of the Thr325Ile single nucleotide polymorphism (SNP) on TAFI antigen levels and potential effects on the severity of SCD in a cohort of Egyptian patients. Genotyping of Thr325lle polymorphism using Taq-Man SNP genotyping assay and TAFI level measurement using an enzyme-linked immunosorbent assay were performed for 80 SCD patients (45 homozygous HbSS, 16 S/β0 and 19 Sβ+) as well as 80 age- and gender-matched healthy control subjects. Plasma TAFI levels were higher in SCD patients with Thr325Ile polymorphism, yet the difference was not statistically significant (p=.204). SCD patients with polymorphic genotypes had a greater number of hospital admissions (p=.03). Ten patients with acute chest syndrome had the homozygous polymorphic genotype (GG), and all patients with pulmonary hypertension had the polymorphic genotype (six were homozygous [GG] and five were heterozygous [GA]). Patients with SCD complicated with pulmonary hypertension showed significantly higher plasma TAFI levels (p=.044). The analysis of Thr325Ile polymorphisms combined with plasma TAFI levels suggests that the analyzed SNP could influence plasma TAFL levels and SCD disease severity and hospitalization rates, which could be predictors for complex disease.

Biophysical chemistry behind sickle cell anemia and the mechanism of voxelotor action

Sickle cell anemia disease has been a great challenge to the world in the present situation. It occurs only due to the polymerization of sickle hemoglobin (HbS) having Pro–Val–Glu typed mutation, while the polymerization does not occur in normal hemoglobin (HbA) having Pro–Glu–Glu peptides. It is also well confirmed that the oxygenated HbS (OHbS) does not participate in the polymerization, while the deoxygenated HbS (dHbS) does, which causes the shape of red blood cells sickled. After polymerization, the blood has a low oxygen affinity. Keeping this fact into consideration, only those drugs are being synthesized that stabilize the OHbS structure so that the polymerization of HbS can be stopped. The literature data showed no systematic description of the changes occurring during the OHbS conversion to dHbS before polymerization. Hence, an innovative reasonable study between HbA and HbS, when they convert into their deoxygenated forms, was done computationally. In this evaluation, physiochemical parameters in HbA/HbS before and after deoxygenation were studied and compared deeply. The computationally collected data was used to understand the abnormal behaviour of dHbS arising due to the replacement of Glu6 with Val6. Consequently, during the presented computational study, the changes occurring in HbS were found opposite/abnormal as compared to HbA after the deoxygenation of both. The mechanism of Voxelotor (GBT-440) action to stop the HbS polymerization was also explained with the help of computationally collected data. Besides, a comparative study between GBT-440 and another suggested drug was also done to know their antisickling strength. Additionally, the effect of pH, CO, CO2, and 2,3-diphosphoglycerate (2,3-DPG) on HbS structure was also studied computationally.

Early Cholecystectomy in Patients with Sickle Cell Disease with Uncomplicated Cholelithiasis Is Associated with Better Outcomes.

Up to 85% of patients with sickle cell disease (SCD) will develop gallstones by their third decade. Cholecystectomy is the most commonly performed procedure in these patients. Cholecystectomy is recommended for patients with SCD with symptomatic cholelithiasis and leads to lower morbidity. No contemporary large studies have evaluated this recommendation or associated clinical outcomes. This study evaluates clinical outcomes after cholecystectomy in patients with SCD and cholelithiasis with specific advanced clinical presentations. The Nationwide Inpatient Sample was queried for patients with SCD and gallbladder disease between 2006 and 2015. Patients were divided into groups based on their disease presentation, including uncomplicated cholelithiasis, acute and chronic cholecystitis, and gallstone pancreatitis. Clinical outcomes associated with disease presentation were analyzed. Statistical analysis was performed using the Student's t -test, chi-square test, ANOVA, and logistic regression. There were 6,662 patients with SCD who presented with cholelithiasis. Median age was 20 (interquartile range 16 to 34) years and 54% were female patients. Cholecystectomy was performed in 1,779 patients with SCD with the most common indication being chronic cholecystitis (44%), followed by uncomplicated cholelithiasis (27%), acute cholecystitis (21%), and choledocholithiasis or gallstone pancreatitis (8%). On multivariable regression, advanced clinical presentation was the strongest predictor of perioperative vaso-occlusive crisis, which was the most common complication. Patients undergoing cholecystectomy for uncomplicated cholelithiasis were at lower risk than those with acute cholecystitis (odds ratio [OR] 2.37; 95% CI 1.64 to 3.41), chronic cholecystitis (OR 1.74; 95% CI 1.26 to 2.4), and choledocholithiasis or gallstone pancreatitis (OR 2.24; 95% CI 1.41 to 3.57). Seventy-three percent of patients with SCD have advanced clinical presentation at the time of their cholecystectomy. After cholecystectomy, perioperative vaso-occlusive events were significantly increased in patients with advanced clinical presentation. These data support screening abdominal ultrasounds and early cholecystectomy for cholelithiasis in patients with SCD.

Impact of Hydroxyurea to Treat Haematological Disorders on Male Fertility: Two Case Reports and a Systematic Review.

Hydroxyurea (HU) is a cytoreductive agent used as standard treatment option for sickle cell anaemia/disease (SCD), essential thrombocythemia (ET), and polycythaemia vera (PV). Despite its overall good safety profile, its use also in relatively young patients raises an interest on its potential impact on spermatogenesis. To perform a systematic review of all published articles investigating fertility in male patients affected by SCD, ET, and PV and treated with HU. Two paradigmatic case reports of patients affected by PV and ET, respectively, have been also reported. PubMed, EMBASE, and Cochrane databases were queried for all the published studies indexed up to November 15th, 2022. A combination of the following keywords was used: "hydroxyurea," "fertility," "male," "sperm," "sickle cell anaemia," "sickle cell disease," "essential thrombocythemia," "polycythaemia vera." Of 48 articles identified, 8 studies, involving 161 patients, were eligible for inclusion. Overall, the number of spermatogonia per round cross section of seminiferous tubule were decreased in patients with SCD compared to healthy males. HU treatment was always associated with a worsening of semen parameters, even up to azoospermia. Notably, treatment discontinuation was associated with an improvement of semen parameters and a trend toward normalization in the case of PV and ET, with a less clear amelioration in men with SCD. In both our patients with either PV or ET, HU discontinuation was associated with a significant improvement of spermatogenesis with successful spontaneous pregnancies. Published evidence do not consistently report normalization of spermatogenesis after HU discontinuation in SCD cases. Conversely, the literature almost consistently reported an improvement of semen parameters at the discontinuation of HU therapy in PV and ET cases. Our real-life two cases confirmed those findings. The willing of fatherhood and the need for effective fertility treatment warrant further research to improve work-up management in men with hematological disorders.

Predicting Hemoglobin Response to Hydroxyurea and Voxelotor in Adults with Sickle Cell Disease

Background: Chronic anemia contributes to morbidity and mortality in sickle cell disease (SCD) and is associated with stroke, leg ulcers, pulmonary hypertension (PH), priapism, and chronic kidney disease (CKD). Treatment options for anemia in SCD are limited - hydroxyurea (HU) is the gold standard treatment, but hemoglobin (Hb) responses to HU in adult and pediatric cohorts appear to be modest at best. We evaluated the Hb response to HU and voxelotor in an adult SCD cohort, as well as baseline factors associated with Hb response to HU. Methods: This longitudinal study was based on retrospective chart review of patients with SCD followed in the UPMC Adult Sickle Cell Program, with drug start dates between 2007 and May 2023. Subjects with a documented start date for HU/voxelotor, a baseline Hb ≤ 3 months prior to starting drug, and a follow-up Hb at 1 or 3 months (± 2 weeks) after the start date were included. Those who self-discontinued therapy were excluded. Baseline factors were collected within ≤ 3 months of drug start date. Given the small number of subjects on voxelotor, factors associated with Hb response were evaluated in the HU group only. Baseline factors were reported using descriptive statistics and compared between Hb responders (defined as Hb increase ≥ 1 g/dL from baseline - a common outcome measure in clinical trials of anti-polymerization agents) and non-responders. Linear regression analysis was performed with change in Hb at 1 month (ΔHb) as the outcome of interest, as Hb levels at 1 and 3 months were highly correlated (r=0.905, p&amp;lt;0.001) and there were fewer missing values for Hb at 1 month. Variables associated with ΔHb with p&amp;lt;0.25 were included in a multivariable regression model. P&amp;lt;0.05 was considered significant, and 95% confidence intervals (CI) were reported. Results: Out of 394 subjects, 93 met eligibility criteria, including 88 starting HU and 5 starting voxelotor. One subject in the voxelotor group was on stable-dose HU. Median ages at start of HU and voxelotor were 24.5 years (range 13-57) and 45.4 years (range 22-60), respectively. Females accounted for 50/88 (56.8%) HU subjects and 4/5 (80%) voxelotor subjects. HbSS genotype made up 47/88 (53.4%) of the HU group and 4/5 (80%) of the voxelotor group. Median baseline Hb in the HU group was 9.8 g/dL, with a median increase of 0.2 g/dL and 0.3 g/dL at 1 and 3 months, respectively. In contrast, baseline Hb in the voxelotor group was 7.2 g/dL with a median increase of 1.9 g/dL and 2.1 g/dL at 1 and 3 months, respectively. Hb response was observed in 21/88 (24%) subjects in the HU group vs. 4/5 (80%) subjects in the voxelotor group. Among responders, median Hb increase was 2.2 g/dL (1 month) and 2.0 g/dL (3 months) with HU. Median baseline ferritin was 200 ng/mL in the HU group and 2413 ng/mL in the voxelotor group. In the HU group, lower baseline Hb was associated with female sex, severe SCD genotype (HbSS, HbSβ 0), and higher HbS%, WBC count, platelet count, and hemolytic markers (lactate dehydrogenase, total bilirubin, and reticulocyte count) (data not shown). Demographic/clinical variables were not associated with Hb response, except for male gender at 3 months (Table 1). Lower baseline Hb, RBC count, and hematocrit and higher MCHC, HbS%, LDH, and total bilirubin were associated with positive Hb response at 3 months. HbF% was higher in the responder groups but was not significant. In a multivariable linear regression model including age, PH, leg ulcers, CKD, baseline Hb, platelets, and total bilirubin as explanatory variables, baseline Hb was negatively associated with ΔHb (B = -0.26, CI = -0.40 to -0.12, p&amp;lt;0.001). History of leg ulcers was also negatively associated with ΔHb but was not significant (B = -0.94, CI = -2.01 to 0.13, p=0.083). Conclusion: Overall, Hb response to HU was much lower compared to voxelotor, though in the 24% of subjects who responded to HU, the Hb increase was comparable. Though limited by small sample size, our data revealed that patients started on voxelotor vs. HU were older, more anemic, and more iron overloaded. Subjects with lower baseline Hb had a more robust Hb response to HU. While limited by the absence of dosing and adherence data, this study provides proof of concept of using baseline characteristics to predict Hb response to SCD therapies, demonstrating the feasibility of a personalized treatment approach in SCD. In addition, our data provide real-world evidence of the effectiveness of HU vs. voxelotor for chronic anemia in SCD.

Persistent Albuminuria and Chronic Kidney Disease in Adults with Sickle Cell Disease: Results from a Multicenter Natural History Study

Background: Patients with sickle cell disease (SCD) are at high risk for developing kidney disease. Albuminuria, an early marker of kidney injury, may be initially transient in patients with SCD. Existing guidelines propose confirming albuminuria at multiple timepoints before intervention. A recent study suggests a urinary albumin-creatinine ratio (UACR) ≥ 100 mg/g correlates highly with persistent albuminuria and subsequent decline in estimated glomerular filtration rate (eGFR). We examined a cohort of patients with SCD to assess those characteristics associated with persistent albuminuria and chronic kidney disease (CKD). Methods During routine clinical visits, we enrolled patients with HbSS or HbSß 0 thalassemia between 18 and 65 years at 4 centers into a prospective study to assess the natural history of kidney disease in SCD. We excluded patients if they had bone marrow transplantation, cancer, connective tissue disease, diabetic nephropathy, other glomerular diseases, hepatitis (B or C), HIV, or were on dialysis. Blood and urine were collected for routine clinical laboratory assessments. Serum cystatin C was measured by commercially available ELISA, and eGFR was calculated using the CKD-EPI-2012 cystatin C equation. We defined CKD as an eGFR &amp;lt; 60 mL/min/1.73m 2 or presence of albuminuria. Glomerular hyperfiltration was defined as an eGFR&amp;gt;130 in females and &amp;gt;140mL/min/1.73m 2 in males, respectively. Persistent albuminuria was considered present if 2 or 3 UACR values were ≥30 mg/g or if initial baseline value was ≥100 mg/g (PMID: 32289161). We utilized generalized linear models adjusting for age and sex to identify characteristics associated with persistent albuminuria and CKD. For multivariable selection, we fit binomial or multinomial LASSO regression models adjusted for age, sex, hemoglobin, %Hemoglobin F, hemoglobinuria, and hydroxyurea to identify those variables associated with outcomes of interest. Results Of 284 enrolled patients, 268 had complete data for UACR, cystatin C and serum creatinine at baseline; 104 (38.8%) of these had persistent albuminuria (Table 1). CKD was present in 40.7% of patients and 26.5% exhibited hyperfiltration. In analyses adjusted for age and sex, several factors were associated with the likelihood of persistent albuminuria. Higher hemoglobin, higher %Hemoglobin F, higher serum bicarbonate and use of NSAIDs were associated with decreased likelihood of persistent albuminuria. Higher systolic and diastolic blood pressure, elevated serum alkaline phosphatase, total bilirubin, and white blood cell count (WBC) were associated with an increased likelihood of persistent albuminuria as were presence of hemoglobinuria and use of angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB). In multivariable selection, higher systolic and diastolic blood pressure, ACEi/ARB use, leg ulcers, higher WBC, higher aspartate transaminase (AST) remained associated with higher likelihood of persistent albuminuria. Presence of avascular necrosis, NSAID use, and higher serum bicarbonate were associated with lower likelihood of persistent albuminuria. When evaluating baseline UACR, individuals with UACR≥100 mg/g had similar probability of subsequent albuminuria as compared to those with severe albuminuria (UACR≥300 mg/g) [Table 2]. Conclusion Persistent albuminuria in this cohort was associated with multiple clinical features. Lower use of NSAIDs in this cohort likely represents counseling of patients to avoid these due to their kidney disease. The relationship with serum bicarbonate suggests a propensity for metabolic acidosis in these patients which could exacerbate the underlying SCD. These data confirm the predictive value of a single UACR value ≥100 mg/g for persistent albuminuria, suggesting this may be a sufficient trigger to warrant intervention. CKD, hyperfiltration and albuminuria were extraordinarily common, consistent with prior studies and validate use of this cohort for additional longitudinal assessment of kidney disease in SCD.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

Addressing Chronic Anemia in Sickle Cell Disease: Characterizing Erythropoiesis-Stimulating Agent (ESA) Utilization and Practice Patterns Among Providers

Background: Chronic anemia exerts profound impacts on the morbidity, mortality, and quality of life in individuals with sickle cell disease (SCD), but therapeutic options are limited. Erythropoietin-stimulating agents (ESAs) are the standard of care for managing anemia in chronic kidney disease (CKD). However, their utilization in SCD remains ad hoc, with limited literature and no guidelines supporting the use of ESAs in SCD. To address this knowledge gap, we present preliminary findings from a survey designed to evaluate provider practices and identify factors influencing the use of ESAs in SCD. Methods: We conducted a cross-sectional web-based survey from March-July 2023 among providers taking care of patients with SCD in the United States (US) and internationally. Survey items were developed to assess provider demographic characteristics, practice environments, and provider practices around ESA use in SCD. The data was collected and managed using REDCap electronic data capture tools hosted at the University of Pittsburgh. Descriptive statistics were used to report the data. Results: The survey has been completed by 33 providers as of a cut date of July 23, 2023. Most providers were hematologists who practiced in an academic setting (23/33, 69.7%), with an equal gender distribution (Table 1). The number of patients with SCD seen annually varied, with 101-300 patients being the most common cohort size (13/33, 39.4%). In the 12 months prior to survey completion, 15/33 (45.5%) providers treated at least 1 patient with SCD with ESAs, usually outpatient (Table 2). Commonly reported barriers to outpatient ESA therapy included lack of insurance approval/coverage or cost concerns, lack of provider experience, and patient tolerability/adherence or inability to perform self-injections. Fewer respondents reported no barriers to prescribing ESAs in the outpatient setting, compared to in the acute/inpatient setting (18.2% vs. 48.5%). The most commonly reported indications for starting ESAs in the outpatient setting were chronic anemia from end-stage renal disease or CKD, reduction of blood transfusion requirement, and chronic anemia in patients refractory to approved therapies. Less commonly reported indications include severe or symptomatic anemia alone and chronic anemia in the setting of cardiopulmonary disease. Respondents reported several contraindications to starting ESAs, including a history of venous thromboembolism (VTE), malignancy, stroke, coronary artery disease, hypertension, and frequent vaso-occlusive crises. Respondents also expressed concerns about the cost/insurance coverage, efficacy, safety, patient burden, and appropriate dosing of ESAs in patients with SCD. Only 18/33 (54.5%) respondents reported ever treating outpatients with SCD with ESAs; nearly all of them used 1 st generation ESAs, and a majority used 2 nd generation ESAs. For 1 st generation ESAs, the most common starting dose was 3500-7000 units (U) (50-100 U/kg; 22.2%) or 7001-14000 U (101-200 U/kg; 22.2 %). Once weekly was the most common dosing interval (50%), followed by three times a week (TIW; 27.8%) and twice a week (16.7%). The maximum dose used was 56001-70000 U (801-1000 U/kg) TIW. For 2 nd generation ESAs, the most common starting dose was 141-210 mcg (2.1-3.0 mcg/kg) (22.2%), and administration often occurred once every 2 weeks (44.4%) or weekly (27.8%). The maximum dose used was 211-315 mcg (3.1-4.5 mcg/kg) weekly. Outpatient ESAs were most often initiated for hemoglobin (Hb) &amp;lt; 6 g/dL (8/18, 44.4%), and Hb &amp;gt; 9 g/dL was the most common treatment target (6/18, 33.3%). ESAs were often combined with hydroxyurea, voxelotor, and simple transfusions (Table 2). Conclusion: These preliminary survey results show considerable variability in how ESAs are currently being used in people with SCD. These variations in clinical practice highlight both significant ambiguity about what works in this setting and the need for further research into the optimal use of ESAs for the management of chronic anemia in SCD (ongoing clinical trial: ACHiEvE-SCD, NCT05451940). Future directions include evaluating differences in practice patterns among adult vs. pediatric and US-based vs. non-US-based providers.

Doping control approach: Identification of equine in vitro metabolites of voxelotor (GBT440), a hemoglobin S polymerization inhibitor.

Sickle cell disease, a debilitating genetic disorder affecting numerous newborns globally, has historically received limited attention in pharmaceutical research. However, recent years have witnessed a notable shift, with the Food and Drug Administration approving three innovative disease-modifying medications. Voxelotor, also known as GBT440, is a promising compound that effectively prevents sickling, providing a safe approach to alleviate chronic hemolytic anemia in sickle cell disease. It is a novel, orally bioavailable small molecule that inhibits hemoglobin S polymerization by enhancing oxygen affinity to hemoglobin. The investigation demonstrated that voxelotor led to an unintended elevation of hemoglobin levels in healthy individuals by increasing serum erythropoietin levels. Voxelotor and its metabolites in an in vitro setting utilizing equine liver microsomes were discussed. Plausible structures of the identified metabolites were inferred through the application of liquid chromatography in conjunction with high-resolution mass spectrometry. Under the experimental conditions, a total of 31 metabolites were detected, including 16 phase I metabolites, two phase II metabolites, and 13 conjugates of phase I metabolites. The principal phase I metabolites were generated through processes such as hydroxylation, reduction, and dissociation. The presence of glucuronide and sulfate conjugates of the parent drug were also observed, along with hydroxylated, reduced, and dissociated analogs. The data acquired will accelerate the identification of voxelotor and related compounds, aiding in the detection of their illicit use in competitive sports. It is crucial to emphasize that the metabolites detailed in this manuscript were identified through in vitro experiments and their detection in an in vivo study may not be guaranteed.

Healthcare Utilization and Cost of Care Analysis of Patients with Sickle Cell Disease with High Vs. Low Biomarkers: A Retrospective Chart Review

Sickle cell disease (SCD) is a red blood cell (RBC) disorder that is associated with frequent and unpredictable vaso-occlusive episodes (VOEs) including pulmonary hypertension, acute chest syndrome, chronic anemia, priapism, splenic and renal dysfunction, and stoke. The total cost to manage a SCD patient with a life expectancy of 64 years is between $1.3-$2.1M. Within the United States alone, up to 67% of adults have ≥3 VOEs per yearthat accounts for most of the healthcare utilization, including 78% of emergency room (ER) visits and 95% of hospitalizations. Despite the same single gene mutation of beta-globin, the frequency of VOEs amongst patients is highly variable. The pathophysiology of VOEs involves a complex interplay between circulating blood cells and elevated plasma factors that promote abnormal interactions to the vascular endothelium. Nearly a half century ago studies demonstrated RBC adhesion strongly correlates with disease severity however only recently standardized testing has become available to assess adhesion clinically. More specifically, we developed a suite of RBC health biomarkers and deployed our flow adhesion bioassays in our landmark, observational study (ELIPSIS). We established that flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA -WB-VCAM) at baseline stratified SCD patients with severe disease phenotypes and validated the clinical predictive value of FA-WB-VCAM two years post-ELIPSIS. The objective of this study was to determine whether baseline FA-WB-VCAM biomarker levels could predict patients more likely to receive direct healthcare contact (ER visits/hospital admissions) to manage SCD complications. This study was a retrospective assessment of clinical FA-WB-VCAM in individuals with SCD during steady state between January 2020 through March 2023. Flow adhesion assays were performed using pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). SCD patients that received at least 3 steady state FA-WB-VCAM biomarker evaluations within 1-year were included in this analysis. Clinical site received an IRB waiver to obtain patient-reported ER visits and hospitalizations during relevant time periods. We calculated a composite adhesion index by averaging baseline FA-WB-VCAM biomarker evaluations and established SCD disease severity by quantifying the number of ER visits and hospital admissions one year after the most recent FA-WB-VCAM biomarker evaluation. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value &amp;lt; 0.05 was considered statistically significant. Out of a total of 3939 clinical tests performed, 366 samples were obtained for “baseline” indications. Of these, only 28 samples were analyzed from 9 unique patients who met the study criteria described above. The average flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM) in SCD patients with high adhesion phenotypes (&amp;gt;400 cells/mm 2) was significantly higher than the low/moderate SCD cohort (mean high=603±78 cells/mm 2, mean low/moderate=343±28 cells/mm 2, p=0.02). More important, the high adhesion cohort had more ER visits (11 vs. 5) and hospitalizations (24 vs 9) and significantly higher healthcare costs ($35,871 vs. $13,563). This is the first healthcare cost analysis assessing the relationship between SCD patients stratified by their adhesive phenotype using the standardized, clinical adhesion biomarker FA-WB-VCAM, and healthcare utilization. The previous ELIPSIS study established that baseline FA-WB-VCAM stratifies patients based on their risk of developing self-reported VOCs during a 6-month longitudinal observation period and their risk of higher clinical disease severity in a 2-year follow-up study. Consistent with this data, the current study demonstrated that a cohort of SCD patients with elevated baseline adhesion phenotypes based on FA-WB-VCAM had a greater utilization of healthcare resources (ER visits and hospitalizations) and had a higher cost of care during the retrospective observation period. These data suggest that RBC health as indicated by “baseline” FA-WB-VCAM levels may predict lower health care utilization and costs, and that this biomarker may be a plausible surrogate endpoint to assess the pharmaco-economic impact of SCD-modifying therapies.

SR-18292 Induces Fetal Hemoglobin Synthesis and Reduces Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a commonly inherited blood disorder, which affects millions around the world. Reactivation of fetal hemoglobin (HbF) synthesis in adult erythroid cells can diminish the severity of many clinical features of the disease. Up-regulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to induce HbF levels in human primary erythroid progenitor CD34 + cells. Here we reported the in vitro and in vivo effects of a small molecular compound, SR-18292, on HbF induction via modulating PGC-1α. To determine whether SR-18292 can induce HbF in human CD34 + cells, we performed RT-qPCR, flow cytometry, and western blot to measure the mRNA and protein levels of PGC-1α and γ-globin. We found that mRNA levels of PGC-1α and γ-globin were significantly increased in cells treated with SR-18292 while β-globin mRNA levels were unaffected. Protein levels of γ-globin and the ratio of F-cells were increased in treated cells. Erythroid cellular differentiation markers (CD71/CD235a) and terminal differentiation markers (CD49d/CD233) were used to examine the differentiation status. There was no significant difference in the population of CD71 +CD235a + cells among all groups. However, SR-18292 treatment increased the percentage of CD49d -CD233 + cells at 5 or 7.5 μM. These results suggest that SR-18292 at higher concentrations can promote erythroid terminal differentiation. To evaluate whether the HbF induction by SR-18292 can reduce the SCD disease pathology in SCD mice or not, we intraperitoneally administered SR-18292 to SCD mice. HbF induction was determined by flow cytometric analysis using an anti-HbF antibody. And it was shown that the HbF-high cell population was induced in SCD mice after 4 weeks of treatment. And we used transferrin receptor CD71 and erythroid-specific marker Ter119 to determine whether SR-18292 could affect erythroid differentiation. There was no significant difference in the CD71 +Ter119 + population after 4 weeks of SR-18192 treatment compared with untreated animals. The mRNA levels of γ-globin and PGC-1α were increased in treated mice. And the increase of PGC-1α and γ-globin protein levels were confirmed by western blot. Reticulocyte is a sign of premature RBC destruction (hemolysis), and the SCD mice usually have high reticulocyte counts (&amp;gt;50%). We measured the reticulocyte in the peripheral blood of SR-18292 treated and untreated SCD mice by flow-cytometric analyses and found that reticulocyte was significantly reduced in SR-18292 treated animals (from 55% to 20%) after 4 weeks of treatment. Complete blood cell counts (CBC) showed that SR-18292 treated mice had slightly elevated numbers of red blood cells (RBCs) and more hemoglobin, but lower counts of white blood cells, lymphocytes, and neutrophils. In addition, the RBC distribution width (RDW) was significantly reduced after SR-18292 treatment, indicating that the size of circulating RBCs was more uniform in treated mice. In addition, erythroid cell morphology was evaluated by blood smears with Wright-Giemsa stain. The number of sickled RBCs were reduced in SCD mice treated with SR-18292 for 4 weeks. This is consistent with our observation that RDW was reduced in SCD mice treated with SR-18292. Furthermore, SR-18292 treated mice had a statistically significant reduction in spleen size and weight in contrast to the spleens of untreated SCD mice. The indices of RBCs were improved in accompany with reduced hemolysis and a higher fraction of circulating mature erythrocytes in treated SCD mice. There were no observable adverse effects on other hematopoietic cell lineages. We also performed single-cell RNA-seqs in human CD34 + cells treated with SR-18292 and found that some known HbF repressors (such as BCL11A and ZBTB7A) were decreased in treated groups. Besides that, there were many differentially expressed genes (DEGs), which might be novel HbF regulators. We are verifying those DEGs genes by qPCR and using CRISPR/cas9 to examine their functions in globin regulation. In summary, our findings suggest that SR-18292 might be a promising therapeutic agent to treat SCD and modulating PGC-1α activity or the signaling pathways that it regulates might benefit therapeutically patients with SCD.

Dietary Behaviors and Social Determinants of Health Among Adults Living with Sickle Cell Disease

Background: Social determinants of health (SDoH) may impact outcomes in sickle cell disease (SCD), and recent literature reports that particular SDoH, such as food insecurity and housing insecurity, may impact diet choices for individuals with SCD. Notably, health outcomes in SCD are linked to diet choices through increased protein and caloric needs, and through nutrients, such as polyphenols, omega fatty acids and micronutrients, which are involved in SCD pathophysiology. The connection between SDoH and diet quality may occur through multiple pathways including access to food, affordability, chronic pain and psychosocial factors at the individual and family level. Importantly, the impact of this connection may relate to outcomes such as pain frequency, hospitalizations, and SCD pathophysiologic biomarkers, such as inflammation. Study Design and Methods: The Diet and SCD Study (NCT: 05170412) is an observational mixed-methods study initiated in 2021 at the National Institutes of Health Clinical Center (NIH CC) in Bethesda MD with a target sample of 75 adults living with SCD in the United States. The purpose of the study is to evaluate associations between SDoH (food insecurity, neighborhood food access, education, employment, healthcare access), dietary intake, and population health related outcomes among adults living with SCD. Study inclusion criteria consists of adults (≥18 years of age) residing within the United States at the time of screening who have a confirmed diagnosis of sickle cell disease with any genotype, and are not hospitalized at the time of enrollment. To ensure participation from adults in diverse regions of the United States, the study has a hybrid design of in-person and telehealth procedures. In-person protocol visits are conducted at the NIH CC. The CC clinical visit includes a clinical assessment, anthropometric measurements, phlebotomy, as well as monitored/proctored electronic surveys that collect SDoH and disease severity variables. The telehealth protocol visit includes a virtual clinical assessment, phlebotomy at a local laboratory center, as well as the same monitored/proctored electronic surveys completed by CC clinical visit participants. A dietary behavior semi-structured interview is conducted at both the CC clinical visit and telehealth protocol visit to garner participants' personal stories related to food choices and diet. Laboratory variables include SCD disease specific markers, amino acid levels, and measurement of inflammatory cytokine markers. All participants undergo two unannounced phone-administered 24-hour dietary recall interviews with trained NIH nutrition staff to measure recent dietary intake. Data from 24-hour recall interviews are used to determine fluid, caloric, macro and micronutrient, and polyphenol intake. Usual dietary intake of foods of potentially related to SCD pathophysiology (cruciferous vegetables and thiocyanate-rich foods) is measured through self-administered electronic 12-month food frequency questionnaires. Primary objectives are to evaluate social determinants of health related to food security and access relevant to disease severity among adults living with sickle cell disease in the U.S. Secondary outcomes are to evaluate the association of dietary quality, specific dietary nutrient intake and serum levels, including amino acids and polyphenols, with disease severity. Tertiary outcomes include evaluating inflammatory and glucometabolic biomarkers with measures of social determinants of health. When completed, the Diet and SCD study will provide important information on the associations between SDoH, dietary quality, inflammatory biomarkers and disease severity for adults living with SCD and provide initial data for future cohort and intervention studies.

A quantum chemistry background of sickle cell anemia and gaps in antisickling drug development

Sickle cell anemia disease has been a great challenge for the world in the present situation. It occurs only due to the polymerization of sickle hemoglobin (HbS) having Pro-Val-Glu (PVG) typed mutation, while the polymerization does not occur in normal hemoglobin (HbA) having Pro-Glu-Glu (PGG) residues. According to data from the literature, Val-beta6 of Pro-Val-Glu is hydrophobic in nature, which appears to fit into a hydrophobic pocket in the adjacent HbS. After the insertion of Pro-Val-Glu into a hydrophobic pocket on the adjacent HbS, the polymerization is started. This is a questionable point on how the replacement of glutamic acid with valine in HbS makes it more reactive to fit into a hydrophobic pocket on adjacent HbS for polymerization. No data from the literature on the reactivity of HbS for polymerization was found yet. This is the first time that the theoretical calculation was done in both HbA and HbS where they were structurally different. After that, a comparative study between PVG and PGG was done at quantum level for the evaluation of the reactivity to fit into a hydrophobic pocket on adjacent HbS. At a quantum level, it was found that the HOMO-LUMO gap of Pro-Val-Glu was lower than that of Pro-Glu-Glu. According to the data from the literature, the lesser HOMO-LUMO gap promotes the initiation of the polymerization reaction. On the basis of the results, it was also shown how the mutation point (Pro-Val-Glu) in HbS becomes more reactive to polymerization, whereas Pro-Glu-Glu in HbA does not. The computational method developed for the first time will be very helpful not only for molecular biologists but also for computational and medicinal chemists. Additionally, the required modifications based on gaps in anti-sickling drug development are also suggested in the presented article.

Plasma Lipid Levels in Relation to Disease Severity in Sickle Cell Anaemia in Abakaliki, Southeast Nigeria

Background: Dyslipidaemia has been implicated in the pathophysiology of sickle cell disease (SCD) complications; hence its role requires further elucidation. Objectives: To investigate the relationship between disease severity and plasma lipid levels of patients with sickle cell anaemia. Methods: A cross-sectional study design was used for the survey. A total of 50 patients with sickle cell anaemia and 50 controls without SCD were recruited for the study. The clinical data and plasma lipid levels of lipids and haemoglobin parameters were analysed. Results: The majority of the participants were aged 18-25 years. Total plasma cholesterol and HDL-C were significantly lower in individuals with SCA compared with the controls (3.3±1.2 vs 4.2±1.2; p&lt;0.001) and (1.3±0.5 vs 1.5±0.4; p = 0.038) respectively. Most patients with SCA had moderate disease severity (24; 48%). There was no statistically significant difference in the plasma levels of total cholesterol and HDL-C across the disease severity groups of SCA (p = 0.694 and 0.262). There was also no significant correlation between total cholesterol, HDL-C, and markers of haemolysis, haemoglobin F, and haemoglobin S levels. Conclusion: SCA is characterised by lower mean plasma TC and HDL than controls. However, no relationship was found between TC, HDL levels and SCD disease severity, markers of haemolysis, HbF and HbS levels. Further studies are required to ascertain the implications of plasma lipid levels in SCD.

Evaluation of liver enzymes activity and some trace elements in sickle cell anemia patients in Basrah Governorate – Iraq.

Sickle cell anemia is one of genetic diseases caused by hemoglobin biochemical disorder in especially in polypeptidic chain of this protein. The study was conducted from October 2019 to December 2020 and included two sets of samples Patient and Healthy .The current study was curried out to evaluate some enzymes and trace elements in patients with sickle cell anemia. The levels of copper (Cu) were estimated spectrophotometrically by reaction of copper with 4 – (3,5 – dibromo – 2- pyridylazo) – N – ethyl sulphopropylaniline to produce a chelating complex.The determination was carried out at wavelength equal to 520 nm. Finally, absorbances were recorded and they are proportional to concentration of copper in the sample . Also, the levels of zinc in serum of blood belonging to sickle cell anemia (SCA) were investigated and estimated by reaction of zinc with chromogen existing in the characteristic kit to produce coloured complex. The absorbances were recorded spectrophotometrically which are proportional to concentration of zinc in blood sample . The levels of biochemical variables were estimated using ARCHITECT c4000 systems provided by ABBOTT Company, which are designed with advanced technology, accuracy in clinical results and ease of handling. The device uses a sample size at a rate of 6µL in all measurements of chemical variables.The total values of the current results were made and expressed by calculation of the mean ± standard deviation. Analysis of all results data were carried out by using a statistical procedure according to analysis of variance univariate . Statistical programmer of social science (SPSS ,v.23 ) was depended for calculation of significant differences for the different groups of patients infected by SCA disease and healthy persons . The correlation regression coefficient (r) was used and depended to distinguish between the means of groups belonging to all patients and healthy individuals ( control group ) .The p-value was agreed for less than 0.05 to the limit of lowest significance . A highly significant difference was recorded belong to levels of GPT, ALP and GOT in patients compared with healthy individuals (control group) according to age and sex factor.

Use of multiple urinary biomarkers for the early detection of chronic kidney disease in sickle cell anemia.

Use of multiple urinary biomarkers for the early detection of chronic kidney disease in sickle cell anemia.

Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Longitudinal Predictors of Pain in Pediatric Sickle Cell Disease.

Despite the identified pathophysiology of vaso-occlusive pain in sickle cell disease (SCD), predictors of pain in youth with SCD remain elusive. In this study, we measured changes in pain frequency, intensity, and interference over 1 year and examined biopsychosocial risk factors (SCD disease severity, age, female, depression, and sleep quality) as possible longitudinal predictors. Medical history was obtained from retrospective chart review for 79 children with SCD (ages 2-18 years; 48.1% female; 100% Black/African American; 83.5% SCD, SS genotype). As part of a clinical screening protocol, caregivers (n = 79) and youth 8-18 years (n = 43) completed psychosocial questionnaires approximately 1 year apart (M = 15.52 months, SD = 5.69). Zero-order correlations, paired t-tests, and hierarchical linear models examined longitudinal predictors of pain. The longitudinal bidirectional relationship between pain and sleep was also examined. The rate of severe SCD disease increased from 41.8% to 55.7% across the year, while most hematologic medical parameters remained stable. Increased depression and pain interference at survey 1 significantly predicted increased pain interference at survey 2. Poor sleep quality and increased pain frequency at survey 1 predicted increased pain frequency at survey 2. Finally, increased pain interference at survey 1 predicted poor sleep quality at survey 2. History of pain, depression, and sleep quality were longitudinal predictors of pain over 1 year in youth with SCD. Identifying longitudinal predictors of pain may lead to earlier identification of patients with a high-risk SCD pain phenotype and earlier medical, psychological, and behavioral interventions.