HbSS Genotype Research Articles

DNA Methylation Changes in Nucleated Red Blood Cells from Patients with Sickle Cell Disease: A Potential Marker of Engraftment after Stem Cell Transplantation

Hematopoietic cell transplant (HCT) is the only established cure for sickle cell disease (SCD). Engraftment (chimerism) of donor red blood cells (RBCs) determines HCT success but current methods measure only white blood cell chimerism and may be inaccurate or fail to indicate graft rejection in a timely manner. Measurement of differentially methylated regions (DMRs) in nucleated red blood cells (nRBCs) to distinguish individuals with sickle cell anemia (genotype HbSS) nRBCs from HbAA and HbAS nRBCs to quantify RBC chimerism could improve post-HCT monitoring by increasing sensitivity to detect rejection and allow for early intervention. Cell-free DNA derived from nRBCs represents a potential method to specifically measure RBC chimerism. Methylation of cytosine nucleotides that are followed by a guanine (methylated CpG sites) is a major means of cell-specific gene expression, and measuring regions of multiple differentially methylated cytosines (DMRs) enables the identification of cell-of-origin of DNA fragments. Cell-free DNA is released into the circulation from all body tissues and cells, including nRBCs, and retains their cell-specific methylation signatures (figure 1). We hypothesized that DMRs unique to healthy and/or SCD nRBC DNA could be identified and later quantified to assess RBC donor chimerism. We generated genome-wide methylomes of nRBC DNA from pediatric SCD (n=2) and non-SCD control (n=3) patients using enzymatic methyl-sequencing (figure 2). From these 5 novel nRBC methylomes and publicly available methylomes and hydroxymethylomes from 35 different tissues and hematopoietic cells we computationally identified 105 DMRs uniquely found in nRBCs compared to other tissues and cells. There were 34 DMRs that specifically distinguished control nRBCs from SCD nRBCs. The 105 DMRs unique to nRBCs were all relatively hypomethylated. These DMRs were intergenic and not associated with CpG islands or shores. DMRs that distinguished SCD nRBCs from control nRBCs were predominantly hypermethylated (76%) in SCD nRBCs and were commonly (21%-24%) located at promoter, exon or intron sequences while 47% were intergenic. 24% were in CpG islands and 5% were in CpG shores. The most significant gene ontology terms in SCD-specific nRBC DMRs were cardiac-related, including heart contraction and conduction. Other highly significant terms were related to cell signaling, transmembrane transporters, and ion channels. Enriched pathway analysis identified terms related to cell signaling, though these were associated with neuronal and immune signaling pathways. To validate our in silico findings, we amplified 8 DMRs from genomic DNA isolated from 23 different cells and tissues and compared methylation patterns. Four DMRs were relatively hypomethylated in control nRBCs compared to SCD nRBCs and other tissues and cells. Amplification of these DMRs from cell-free DNA in patient plasma before and after HCT will validate them as potential unique markers of successful RBC chimerism. Quantification in recipients with HbAA and HbAS donors will be performed. The successful identification of nRBC-specific DMRs in plasma cell-free DNA will enable novel methodology for the quantification of RBC chimerism after HCT for SCD that can be compared to conventional methods of chimerism measurement in future clinical trials.

Plasma Proteome Profiling of Sickle Cell Patients Reveals Activation of Inflammatory Pathways during Steady State and Vaso-Occlusive Crisis

Background The pathophysiology of Sickle Cell Disease (SCD) is characterized by chronic intravascular hemolysis and ischemia-reperfusion, resulting in oxidative stress and chronic inflammation. Although inflammation seems to play an important role in the development of tissue damage in SCD, little is known about the various cellular and cytokine pathways involved in the pathophysiology of SCD. This study aims to further elucidate the inflammatory pathways in SCD using targeted plasma protein profiling both in steady state and during vaso-occlusive crisis (VOC). Methods In this prospective study, SCD patients with the genotypes HbSS and HbSβ 0-thalassemia and healthy ethnicity matched controls were included. Plasma levels of 92 proteins involved in various inflammatory pathways were measured in EDTA plasma using Olink Proximity Extension Assay (PEA) technology. In brief, pairs of oligonucleotide-labeled antibody probes were added to 20 μL of plasma, resulting in the binding of these antibody probes to their targeted protein. Subsequently DNA polymerase was added, leading to a proximity-dependent DNA polymerization event, generating a unique PCR target sequence. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, which is an arbitrary unit on a log2-scale with an NPX difference (estimate) of 1 meaning a doubling of protein concentration. RStudio version 4.2.1 was used for data analysis and visualization. Differences between subgroups were tested using an unpaired or paired t-test as appropriate. Results were corrected for multiple testing using the Benjamini-Hochberg method. Results with p < 0.05 were considered significant. Furthermore Over-representation Analysis (ORA) was performed to investigate the association of the differentially expressed proteins with specific biological processes. Results Plasma samples were collected from 43 SCD patients in steady state (median age: 25 years (IQR: 20 - 32)) and 13 healthy ethnicity matched controls (median age: 29 (IQR: 28-39)). From 15 SCD patients plasma samples were collected during both steady state and VOC (median age: 27 (IQR: 19 - 34)). Comparison of protein levels between healthy controls and SCD patients in steady state identified 38 proteins (IL-18, ANGPT2, Gal-9, CD4, HGF, HO-1, CCL4, ARG1, ADA, GZMA, PD-L1, CCL23, LAG3, TNFRSF4, CCL3, CXCL11, CRTAM, IL-10, GZMB, CD27, FGF2, IL-8, IL-6, TNF, PGF, NCR1, TNFSF14, CASP-8, IL-7, FASLG, TNFRSF9, LAP TGF-beta-1, PD-L2, CCL19, CD83, GZMH, MCP-4, CD244, TIE2) that were significantly higher in SCD patients as shown in Figure 1. Furthermore soluble CD70 was significantly lower in SCD patients in steady state compared to healthy controls (estimate: -0.34, p < 0.05). Over-representation Analysis revealed a significant upregulation of proteins involved in the regulation of hematopoiesis, the IL-18 signaling pathway, T-cell activation, leukocyte differentiation and leukocyte adhesion (Figure 2). Pairwise comparison of protein levels between steady state and VOC in SCD patients revealed 6 proteins (TWEAK, VEGFR-2, CD244, PDCD1, GZMA, TNFRSF4) which were significantly lower and 12 proteins (IL-6, TNFSF14, ADA, IL-15, HGF, IL-10, IL-8, Gal-9, CSF-1, CCL20, MMP7, VEGFA) which were significantly higher during VOC as compared to steady state. During VOC, pathways involved in the regulation of cell death (IL-6, TNFSF14, ADA, HGF, IL-10, PDCD1, VEGFA) and tissue development (IL-6, ADA, HGF, IL-10, VEGFA) were significantly enriched. Summary/conclusion The results of these proteomic analyses provide new insights into the pathways that regulate inflammation in SCD, both during steady state and VOC. These findings might contribute to a better understanding of SCD pathophysiology and identifying potential new targets for therapeutic interventions in SCD patients. Our results warrant further in-depth investigation of the different activated pathways.

Fruit and Vegetable Intake in Adults with Sickle Cell Disease

Introduction Patients with sickle cell disease (SCD) experience frequent disruptions to their daily lives from vaso-occlusive pain episodes and chronic organ complications, such as stroke. These acute and chronic complications negatively impact patients' ability to continue their educational and occupational obligations, making them vulnerable to socioeconomic challenges (PMID 30540112). In children with SCD, individual-level socioeconomic distress was associated with poor diet quality (PMID 34811867). In the non-SCD literature, food insecurity is characterized by a low fruit and vegetable diet intake and a greater risk of chronic kidney disease (PMID 24434743). The association of individual level socioeconomic distress with fruit and vegetable intake and, in turn, of fruit and vegetable intake with kidney function in adults with SCD is unclear. Methods We prospectively surveyed adults with SCD to investigate the association between individual level socioeconomic distress, fruit and vegetable intake, and kidney function. Between July 2022 and June 2023, we enrolled 81 adults with a diagnosis of SCD from an outpatient clinic at a Sickle Cell Center in an urban academic institution. We assessed fruit and vegetable intake using the National Cancer Institute's All Day Screener, a validated tool that estimates daily intake of fruit and vegetables. Markers of socioeconomic distress were measured using a modified short quantitative survey created by adapting the Centers for Disease Control's Behavioral Risk Factor Surveillance System Questionnaire. The distressed community index (DCI), a composite indicator that estimates the economic well-being of a community, was determined for each patient's residential address. The data were analyzed using descriptive statistics, and the association of fruit and vegetable intake with body mass index and kidney function was determined using linear regression, adjusting for age, sex, and SCD genotype. Results The median age of the cohort was 38 years (interquartile range [IQR], 31 - 45 years), 60% were female, 74% had Hb SS or Sβ 0-thalassemia genotype, and 47% were on hydroxyurea therapy. The median BMI of the cohort was 25 kg/m 2 (IQR, 21 - 30 kg/m 2). With respect to socioeconomic indicators, 33% (25/75) had high school or less education, 80% (59/74) did not own their residence, 48% (37/77) were unemployed or unable to work, and 71% (49/69) had income of < $35,000/year. The median DCI for our cohort was 90.8 (IQR, 68.9 - 94.9) with 65% (52/81) living in a distressed community (DCI > 80). The median fruit and vegetable servings consumed by the cohort was 1.95 serving sizes per day (IQR 1.05-3.04 servings), which is lower than the Centers for Disease and Control (CDC) Prevention daily dietary recommendation of 5 servings per day, and below the national and state-level (Illinois) average of 2.6 servings per day in the general population (PMID 34990439). Only 15% (11/75) of our cohort met the CDC daily recommendations of fruits and vegetables. Baseline demographics, including age, sex, SS/Sβ 0-thalassemia genotype, and hydroxyurea therapy use, were not different between those that met versus did not meet the daily fruit and vegetable intake requirements (Table). Similarly, we did not observe any differences in the socioeconomic indicators, DCI score, or proportion of patients living in a distressed tier in these two groups. On multivariable analyses, lower fruit and vegetable intake was associated with higher body mass index (natural log β 0.08 ± 0.03; P = 0.001) and higher urine albumin to creatinine ratios (natural log β 0.41 ± 0.2; P = 0.045). We did not observe any association of fruit and vegetable intake with systolic or diastolic blood pressure or with estimated glomerular filtration rate. Discussion In this prospective, cross-sectional study, adults with SCD experienced a high burden of socioeconomic distress. Although we did not observe an association between socioeconomic indicators and fruit and vegetable intake, only 15% of our cohort met the daily fruit and vegetable intake recommendations. Lower fruit and vegetable intake was associated with higher body mass index and albuminuria. Our findings highlight the need for further investigation to better understand barriers to a healthy diet in people with SCD. Furthermore, strategies to increase fruit and vegetable intake may represent a modifiable risk factor to improve SCD-related health.

Development of a Genetic Risk Score for Albuminuria in Pediatric Patients with Sickle Cell Anemia

Introduction Sickle cell anemia (SCA) causes progressive, age-dependent, kidney damage. Albuminuria is an early clinical biomarker for chronic kidney disease (CKD) and is associated with progressive glomerular injury. Identifying patients at risk for albuminuria is critical for early institution of renoprotective interventions to prevent progressive kidney injury. Developing a genetic risk score (GRS) to predict albuminuria is one approach to identifying high risk patients. Our prior data demonstrates that Apolipoprotein-L1 ( APOL1 G1/G2) is associated with early development of albuminuria in children with SCA (PMID:30890594). However, this gene variant does not account for all kidney disease progression. Multiple other gene variants that modify SCA-severity have been associated with CKD in adults with SCA. We hypothesized that developing a genetic risk score (GRS) will allow for identification of pediatric patients at increased risk for development and progression of kidney disease. We aimed to develop a novel pediatric GRS that can identify pediatric SCA patients at high risk for albuminuria. Methods Gene allele variants in APOL1 G1/G2, HMOX1, BCL11A and α-thalassemia (α -3.7 allele), were identified in 324 African American patients with SCA (HbSS or HbS 0-thalassemia genotypes) enrolled in the Sickle Cell Clinical Research and Intervention Program (NCT02098863), a lifespan, longitudinal cohort study. The first urine albumin to creatinine ratio (ACR) collected after birth was analyzed as a continuous variable and dichotomized by a threshold of ACR ≥30mg/g creatinine as albuminuria. We defined a GRS 3 based on established risk alleles in three genes: APOL1 (G1/G2 ), BCL11A (rs1427407), and α -3.7(PMID:27658436). High risk was defined as patients with APOL1 (G1/G1, G2/G2 or G1/G2), without α -3.7 and with wildtype BCL11A (G/G); low-risk was defined as patients negative for APOL1 G1/G2 and with α -3.7 (either α -3.7/αα or α -3.7/α -3.7) and the BCL11A T allele (either G/T or T/T). All other combinations were defined as intermediate risk. By adding HMOX1 (rs743811), C allele (TT/TC vs CC), we further refined GRS 3 as GRS 4 using the summation of the number of high-risk alleles of kidney outcomes for four variants and created four risk categories. The generalized linear regression model was used to associate additive dose-effect models for GRS 3 and GRS 4 with continuous ACR and albuminuria, adjusted for age, sex, hydroxyurea treatment, 5 principal components, and, if significant, the age-genotype interaction. ACR was log-transformed to approximate normality. As we included variants with known associations, we used a one-sided p-value for testing associations of individual variants, GRS 3, and two-sided p-values for GRS 4. P-values <0.05 were considered significant. Results In 324 individuals with SCA, those with albuminuria (34, 10.5%) appeared to be older than those without albuminuria (290, 89.5%) (mean 9.8 years [standard deviation ±4.4 years] vs. 8.6 [± 4.0], p=0.053). There were no significant differences between these groups by sex (female 58% vs. 49%, p=0.37), proportion on hydroxyurea (62% vs. 59%, p=0.85) or fetal hemoglobin (15.6 [± 10.61] vs. 14.8% [± 10.0], p= 0.81). All four individual genetic variants were associated with the first ACR (all p ≤ 0.028) and albuminuria (all p≤ 0.03). GRS 3 was significantly associated with the first ACR (estimate=1.4, standard error [SE]=0.5, p=0.0035) and albuminuria (odds ratio [OR]=3.0, 95% CI: 1.2-7.5, p= 0.008). After adding HMOX1 to GRS 3, GRS 4 was more significantly associated with the first ACR (estimate=0.9, SE=0.3, p=0.0005) and albuminuria (OR=2.0, 95% CI: 1.2-3.4, p=0.003). Summary We developed a GRS 3 based on three genetic variants associated with albuminuria in pediatric patients with SCA and stratified patients into different risk categories. Addition of a fourth genetic variant to create GRS 4improved the model. Our findings will need to be validated in larger longitudinal studies to further examine the association between GRS 4 and development of albuminuria and chronic kidney disease. Genetic prediction strategies using GRS has the potential to identify high risk SCA patients for whom renoprotective measures could prevent the development of albuminuria.

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<0.001) and there were fewer missing values for Hb at 1 month. Variables associated with ΔHb with p<0.25 were included in a multivariable regression model. P<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<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.

Use of Machine Learning to Predict 30-Day Reutilization of Care for Patients with Sickle Cell Disease Treated for Vaso-Occlusive Crisis

Background Sickle cell disease (SCD) is an inherited disorder of red blood cells affecting millions of people worldwide. SCD is characterized by recurrent episodes of severe pain attacks, also called vaso-occlusive crisis (VOC), and are the most common reason for hospitalization. Approximately 90% of the hospital admissions are for pain treatment and the hospital readmission rate is alarmingly high, as 30-50% are re-admitted within 30 days. We previously were successful in developing machine learning models to predict pain using inpatient vital signs data as well as Apple Watch data in patients with SCD hospitalized for VOC. We now aimed to predict reutilization of care within 30 days in patients with SCD treated for a VOC. Methods Patients with SCD aged 18 years and above, who were admitted for a VOC to the day hospital or Duke University Hospital between April and June 2022, were eligible for this study. Following informed consent, demographics, SCD genotype, details from the hospitalization such as length of stay, pain scores, as well as vital signs measured per standard of care were collected from the electronic medical records including blood pressure, pulse rate, respiratory rate, oxygen saturation and temperature. Baseline vital signs during regular visits were collected 6 months before and after the hospitalization. We used the values of nearest neighbors to fill in the empty entries. The vital signs over the whole hospital stay were averaged and those numbers were used as the predictor values for the model. The primary outcome was reutilization of care defined as readmission within 30 days to the day hospital and/or hospital. The predictors were used to fit 3 different machine learning classification models for the prediction of reutilization of care: random forest, logistic regression, gradient boosting. By fitting random forest model on the whole dataset, we were able to rank all the features using mean decrease in impurities. To avoid overfitting, we only used the best four predictors which were diastolic blood pressure, pulse rate, respiratory rate and pain score.The performance of the machine learning models was evaluated using the following metrics: accuracy, precision, recall, F1 score and area under the receiver-operating-curve (AUC). Results Eighteen participants with SCD were included in this study. The median age at inclusion was 30 years (IQR 22-34). The majority of the participants had SCD genotype HbSS (68%), and all were Black of African-American. There were 10 participants treated at day hospital (56%), while the other 8 participants were admitted to the hospital with a median length of stay of 7.5 days (IQR 2.5-10). After discharge, 15 participants sought medical care at least once within 30 days (83%); 8 were hospitalized (44%), and 13 were readmitted to the day hospital (72%). This pilot study consisted of 88 vital sign data points across the 18 patients. The metrics of our best-performing machine learning model, the random forest model, were: accuracy 70%, precision 0.94, recall 0.71, F1 score 0.81 and AUC 0.61 (Figure 1 and 2). The difference in precisions and recalls for all the models reflects the class imbalance in the dataset. To test how the model will perform for independent data sets, we used 5-fold cross-validation, and the cross-validation accuracy was 66% with a standard deviation of 7.7%. Conclusion In this pilot study, our machine learning model was able to accurately predict health care reutilization within 30 days following discharge, with real-time vital signs data collected during clinic visits and hospital admissions in participants with SCD. Prediction of reutilization may help healthcare providers identify those at high risk and allow considerations for inpatient and outpatient strategies for patient management. Our next efforts include prediction of re-utilization from data collected from consumer wearables and in a larger number of participants.

Going Going Gonad: Assessing Iron Staining Impact on Fertility for Patients with Sickle Cell Disease

Introduction: Medical advances in the care of patients with sickle cell disease (SCD) and other hemoglobinopathies have allowed for the focus of care to shift and incorporate long-term quality of life metrics, such as fertility. Concerns for reproductive health in patients with SCD stem from the underlying disease itself as well as from medical therapies that are offered such as hydroxyurea, chronic red blood cell transfusions, and stem cell transplant (SCT). Iron deposition from chronic transfusions in other tissues is known to result in end organ damage and lifelong consequences. While little is known about iron overload and fertility, one study noted testicular iron deposition on MRI in pubertal males with transfusion-dependent beta-thalassemia; results were notable for lower sperm concentrations and a lower percentage of sperm with normal morphology, indicating that iron overload likely has an impact on fertility for patients. Iron deposition has not been studied in the pre-pubertal hemoglobinopathy population. Fertility counseling in this patient population does not often occur until patients are considering SCT as a curative therapy option, as the conditioning regimens for SCT are gonadotoxic. Without a good understanding of the impact of chronic transfusions on fertility for patients with hemoglobinopathies, it is difficult to determine the optimal time to offer fertility preservation to families. We aim to assess iron overload on testicular tissue to gain a better understanding of the impact that medical management and chronic transfusions can have on fertility for a patient with SCD. Methods: Our study is a cross-sectional evaluation of patients with SCD who underwent testicular tissue cryopreservation (TTC) as a part of a multicenter research protocol through the University of Pittsburgh Medical Center (UPMC) prior to SCT. As part of the TTC protocol, each patient provided a small piece of tissue for research, with the remaining cryopreserved for patient use. All samples were evaluated for the presence of germ cells. For this study, demographics, treatment data, imaging results, and labs pertinent to hormonal profile and iron status were obtained. All samples from patients with SCD and thalassemia will undergo additional pathology review to evaluate for the presence of germ cells and iron. At the time of this abstract, the tissue for two patients had been analyzed. Results: Banked research tissue from UPMC includes 77 patients with SCD and 21 patients with beta thalassemia major. Of the 77 SCD patient samples, 42 were found to have germ cells, 34 have not yet been analyzed and 1 had no germ cells present. Of the 21 samples from patients with beta thalassemia major, 17 were found to have germ cells, 3 were not yet analyzed and 1 had no germ cells present. To date, we have obtained pathology review on two patients, both with Hb SS genotype and were on the maximum tolerated dose of hydroxyurea prior to TTC. Patient 1 had been on hydroxyurea for 7 years and had 13 units of blood transfusions, and Patient 2 on hydroxyurea for 4 years and 6units of blood transfusions prior to TTC. Both patients were found to have normal testicular tissue histopathology with presence of germ cells and without evidence of iron on staining (Table 1). Conclusions: Based on our preliminary data it can be noted that patients without significant iron burden, based on low ferritin and a limited transfusion history may not have a significant impact on their testicular histopathology with their iron staining being negative. We will expand our patient cohort to include patients who have a severe disease course requiring more blood transfusions to determine whether this is associated with increased iron deposition in their testicular tissue and/or reduced germ cell numbers. This information will allow teams to optimize the timing of fertility discussions and possible preservation in this patient population, even outside of curative treatment.

Inclacumab Reduce Preexisting Red Blood Cell Adhesion to Activated Endothelial Cells: In-Vitro Assessment of the Microfluidic Platform Endothelium-on-a-Chip

Background: In 2019 FDA approved the use of an antibody anti P-selectin (Crizanlizumab) for the treatment of sickle cell disease (SCD) to reduce the vaso occlusive crisis (VOCs). New anti-adhesive treatments are being developed recently. Global Blood Therapeutics, Inc., acquired by Pfizer Inc. in October 2022, acquired Inclacumab from Roche and started two clinical trials now in phase 3 to evaluate the capacity of this humanized IgG4 monoclonal antibody anti P-selectin to reduce the VOCs in patients with SCD. In our recent work presented at EHA 23 in Frankfurt Germany, we showed that Inclacumab prevents the adhesion of RBCs to chronically and acutely heme activated human umbilical vein endothelial cells (HUVECs) with a statistically significant higher effect in comparison to Crizanlizumab. In this work we used a similar approach to test the capacity of Inclacumab in the reduction of preexisting adhesion events on acutely heme activated HUVECs. Methods:Whole blood EDTA samples from six SCD subjects all with HbSS genotypes were collected at University Hospital Cleveland Medical Center, Cleveland, OH, USA. To standardize the samples, we isolated RBCs by centrifugation and subsequential washes in Phospahte buffer s of the whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. HUVECs (Lonza, Morristown, NJ, USA) were introduced and maintained within the endothelialized microfluidic channels at physiological flow for at least 48-72 hours prior to experiments. We performed an acute short-term activation, where the blood samples were supplemented with 40 µM heme +/- 100 µg/ml injected through the microfluidic channels for 15 minutes, after the adhesion took place we washed the non-adherent cells with Inclacumab and Crizanlizumab and we compared with untreated washing buffer (both compounds were provided by Global Blood Therapeutics, Inc., a wholly owned subsidiary of Pfizer Inc. South San Francisco, CA, USA) A phase-contrast images of the remaining RBCs were acquired with an inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and quantified based on previous published methods (Figure 1A). Paired t-test was used to calculate statistical significance. Results: Acute short-term heme activation induces high level of RBCs adhesion. Both compounds Inclacumab and Crizanlizumab showed a reduction of the number of adherent RBCs to the activated heme, but only Inclacumab results statistically a significant higher reduction compared to untreated RBCs (Figure 1B). Conclusions: Inclacumab is able not only to prevent the adhesion of RBCs to acutely and chronically activated HUVECs but is able to remove the preexisting adhesion and results in significant higher reduction compared to untreated. RBCs adhesion assay on Endothelium-on-a-chip is a novel and efficient way to study the efficacy of the new antiadhesive drug and could be used to monitor patient response to new therapies in SCD. Figure1 : Effect of Inclacumab vs Crizanlizumab on removal of SCD RBC adhesion to heme activated HUVECs. A) Schematic of adhesion experiment and inverted microscope contrast of phase images 10X of adherent RBCs at acutely heme activated HUVECs. B) Inclacumab has statistically significant effect on remove adherent RBCs to acutely heme activated HUVECs (p=0.009, N=7) For all experiments, the heme concentrations were 40 µM and Inclacumab and Crizanlizumab were 100 µg/ml. Scale bare 20 µm. P-values were based on paired t-test. Error bars represent the standard error of the mean (SEM)

A Comprehensive Echocardiographic Portrait of Adult Sickle Cell Disease Patients in the Modern Era

Background Cardiovascular complications, especially pulmonary hypertension, are a known cause of mortality in patients with sickle cell disease (SCD). With advances in medicine and the introduction of various disease-modifying medications, the incidence and prevalence of cardiovascular complications are poorly documented. Aim To determine rates of cardiovascular complications in patients with SCD in the modern era of disease-modifying medications in a single center. Method We performed a retrospective observational single-center study of patients with SCD: all genotyped and ≥16 years old who had a recent echocardiogram performed during the study period of 2019-2023. Demographics, echocardiogram results, and laboratory findings were collected and analyzed. Descriptive statistics are presented in percentages, medians, and interquartile ranges. An unpaired Student's t-test and Fisher's exact test were used to determine, where appropriate, the level of significant differences among continuous and categorical variables, respectively, with p value <0.05 considered statistically significant. Data was analyzed using GraphPad Prism version 9.0.2. Results As of June 2023, 80 patients (38 male and 42 female, median age 30 years) had undergone a routine transthoracic echocardiogram. Sixty-five patients had the HbSS genotype, 8 HbSC, and 7 HbSB null. Sixty-one patients had received at least one treatment, and 19 (24%) were on concurrent therapy. Hydroxyurea was the common SCD-modifying therapy that the patients received (51 patients), followed by L-glutamine and voxelotor (11 each) and crizanlizumab (9 patients). In total, 56 (70%) patients had at least one cardiac abnormality. The most common cardiac abnormality was left atrial dilatation (41%), followed by left ventricular hypertrophy (33%), right atrial dilation (24%), and left ventricular dilatation (23%). Pulmonary hypertension was present in 30 (38%) patients with tricuspid regurgitation velocity (TRV) ≥2.5 msec (Table 1). The cohort of patients with pulmonary hypertension tended to be older (median age 31 years, p=0.0357), have a higher creatinine level (median 0.8, p=0.0414), and have coexisting echocardiographic abnormalities such as left atrial dilatation (70% vs. 24%, p<0.0001) or right atrial dilatation (40% vs. 14%, p=0.0136) than SCD patients without pulmonary hypertension (Table 2). Conclusion We present a comprehensive echocardiographic assessment of patients with SCD. Despite advances in the treatment of SCD, there remains a high prevalence of cardiac involvement in SCD patients. We found out that 70% of the patients we reviewed had at least one cardiac abnormality on echocardiography. Left atrial dilatation and left ventricular hypertrophy were the most common cardiac abnormalities noted. Pulmonary hypertension was observed in 37.5% of patients with co-existing right and left atrial enlargement. Larger studies are needed to corroborate these findings.

Primary Analysis of Spartan: A Phase 2 Trial to Assess the Efficacy and Safety of Crizanlizumab in Patients with Sickle Cell Disease Related Priapism

Introduction: Priapism is a painful, sustained penile erection that occurs in 35% of adult men with sickle cell disease (SCD) and may result from the underlying mechanism of vaso-occlusion. To date, there is no effective disease-modifying drug proven to treat priapism. Crizanlizumab is a monoclonal antibody that binds and blocks P-selectin, a key mechanistic component of vascular endothelial adhesion. Based on the results of the SUSTAIN trial, crizanlizumab was approved by the FDA to reduce the frequency of vaso-occlusive crises in patients with SCD aged ≥16 years. In this primary analysis of the Phase 2 SPARTAN trial (NCT03938454), we evaluated the efficacy and safety of crizanlizumab after 26 weeks of treatment in patients with SCD-related priapism. Methods: The SPARTAN trial included male patients aged ≥12 years with all SCD genotypes who experienced ≥4 priapic episodes lasting ≥60 min over the 14-week period preceding screening. Eligible patients also experienced ≥3 priapic episodes during the subsequent 12-week screening period with ≥1 event occurring within 4 weeks prior to the first treatment. After enrollment priapic events were documented through electronic self-reporting tools and validated patient-reported outcomes surveys for 26 weeks and included start/end date, sleep status, duration, non-pharmacological treatment, triggers, emergency department visit, and pain intensity during the screening period. Baseline priapic events were collected for each patient during the 12-week screening period. Patients received crizanlizumab 5.0 mg/kg IV infusion on week 1, week 3, and every 4 weeks thereafter. The primary endpoint was percent reduction from adjusted baseline period in priapic events by 26 weeks. Percent reduction from baseline in priapic events was tested using a one-sided Wilcoxon's Sign Rank test. Hodges-Lehmann estimate of median percent reduction in priapic events (with 95% CIs) was also reported. Results: Data cutoff was March 28, 2023. Of the 36 patients enrolled, 26 (72.2%) were between 18 and 40 years, and the median (range) age was 29 (14-58) years. Most patients were HbSS genotype (n=29, [80.6%]) and more than half were on hydroxyurea (n=21, [58.3%]). Patient-reported history prior to crizanlizumab treatment revealed that more than half of patients experienced priapism daily or every other day (n=21, [58.3%]) and most reported a duration of 1 to 5 h (n=33, [91.7%]). The most common patient-reported emotions at the start of the study were frustration and exhaustion, reported by ≈50% of patients. The median (IQR) baseline number of priapic events adjusted for 26 weeks per patient was 42 (13-73) events ( Table). All patients experienced events lasting 1 to ≤4 h, although some also experienced severe events lasting >4 to 6 h (n=19, [52.8%]) and >6 to ≤12 h (n=11, [30.6%]). In one case, 38 days of screening period baseline data was not reported, resulting in a strong outlier that affected the results ( Patient 1, Figure). Thirty-six patients received ≥1 dose of crizanlizumab, with a median cumulative dose of 35.0 mg/kg over 26 weeks; 5 patients each missed 1 infusion and 1 patient missed 2 infusions. The Hodges-Lehmann estimate of median percent reduction from baseline in priapic events per patient was 46.0% (95% CI: 23.5%-65.4%; P=.068). Twenty-eight patients (77.8%) reported a reduction from baseline in priapic events ( Figure). A subgroup analysis revealed a trend towards a larger percent reduction in events for patients with ≥22 baseline events, possibly indicating that patients with severe disease benefitted more from treatment. The most frequent treatment-emergent adverse event (TEAE) was headache (16.7%). No patients discontinued crizanlizumab prematurely and only one patient withdrew consent by week 26. Conclusions: Primary analyses from the SPARTAN trial showed that patients with SCD-related priapism treated with crizanlizumab over 26 weeks experienced approximately half as many priapic events compared with baseline. There was a trend towards a more significant reduction in frequency of episodes in patients with severe disease. Crizanlizumab was safe and well tolerated, with observed TEAEs consistent with the safety profile seen in other clinical trials. Final analyses of the SPARTAN trial will provide data over 52 weeks and may provide further support for the role of crizanlizumab in reducing frequency of priapic events.

Predicting Vaso-Occlusive Crises in Sickle Cell Disease through Digital, Longitudinal Tracking of Wearable Metrics and Patient-Reported Outcomes

Background Sickle Cell Disease (SCD) is a genetic disorder caused by an HBB gene mutation and resulting structural variant haemoglobin (HbS) that polymerises upon deoxygenation, leading to a sickled morphology of red blood cells ( Chakravorty et al., Arch Dis Child 2015). This causes acute pain episodes, known as vaso-occlusive crises (VOC), that can result in severe end-organ damage in the long-term and a shortened lifespan ( Jang et al., J Transl Med 2021). Responsible for an estimated 95% of SCD hospitalisations and as a key predictor of death ( Darbari et al., Eur J Haematol 2020, Ballas et al., Am J Hematol 2005), it is critical to find new ways to drive early detection of VOCs in order to support preventative interventions. Aims This work aimed to develop a machine learning (ML) algorithm capable of predicting the potential onset of a VOC, using physiological data captured longitudinally by a wearable smartwatch, as well as patient-reported outcomes (PROs) entered via a specialised mobile phone application. Methods Following informed patient consent, participants gained access to a mobile phone application, or “digital patient wallet”, encompassing their health data and access to a PRO entry portal ( Summers et al., HemaSphere 2023). This allowed for daily recording of PROs including EQ-5D-5L, pain, mood, and fatigue scores, alongside self-reported VOCs. Participants were also provided with a Withings ScanWatch to be worn day and night, capturing physical activity, sleep quality, and heart rate data in an automated manner. Medical record data including healthcare utilisation, pathology, and demographic data was obtained through participant completion of a Subject Access Request form. Data for a snapshot cohort of 186 patients over a 1-month period (May-June 2023) were analysed through an ensemble of ML models: gradient boosting machine, neural network, and k-means clustering. All collated metrics were fed into the model, with the exclusion of hospitalisation as an 'a posteriori' variable, to ensure predictions were based on appropriate variables and not strengthened by self-confirming factors. The resulting predicted likelihood of a VOC for each participant was calculated on each day and given as a scale from 0-100%, with a likelihood of 75% or higher deemed to be a predicted VOC. This was then assessed against the true incidence of the participants' self-reported VOCs. Results The mean age was 36 (SD 11.6) years, and most patients were female (70%). The HbSS genotype was most common (75%), followed by HbSC (16%), and HbS Beta + Thalassemia (5%). 82,804 datapoints were collated for all participants across this period, encompassing 90 different variables. 69 instances of a VOC were self-reported by patients during the period of analysis, 58 (84%) of which were accurately predicted by the algorithm. 2,366 instances of no VOC were self-recorded, giving a negative predicted value of 83% (1,958/2,366 predicted). Further breakdown by the predicted likelihood score is displayed in Figure 2, highlighting the spread of individually calculated VOC likelihood scores per day against the incidence of patient-reported VOCs. Summary/Conclusions This work provides an important translation of daily trackable metrics through a remote, digital ecosystem, to the real-world prediction of potential VOC events in SCD. The purpose of this algorithm is, at its core, to act as a pre-emptive warning system from which proactive behavioural changes or interventions can be enacted. As such, the prediction of additional potential risk scores above the 75% threshold reflects an approach of ‘over-predicting’ considered to be a preferable strategy for the algorithm, given the severe nature of VOCs and the health impacts of missing a potential event. Our ongoing aim is to further develop the algorithm and its contributing metrics against medical data, in addition to patient self-reported events, to further optimise the predictor. This includes the curation of core metrics requiring minimal patient input, in order to reduce missing datapoints across those that feed into the algorithm and thereby reduce unpredicted VOCs. Ultimately, we hope to provide an early alert system that will help patients to identify their VOCs sooner, and thus action the next steps that may prevent possible hospital admissions.

Preliminary Results from a Multicenter Phase 2/3 Study of Next-Generation HbS Polymerization Inhibitor GBT021601 for the Treatment of Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a lifelong inherited disorder resulting in polymerization of sickle hemoglobin (HbS) and is characterized by chronic hemolytic anemia, vaso-occlusive crises (VOCs), and cumulative end-organ damage. Voxelotor is a first-in-class HbS polymerization inhibitor approved in the US for the treatment of patients with SCD aged ≥4 years and in Europe for the treatment of hemolytic anemia due to SCD in patients aged ≥12 years. GBT021601 is a next-generation HbS polymerization inhibitor with improved pharmacokinetic (PK) properties that increases hemoglobin (Hb)-oxygen affinity and stabilizes Hb in the oxygenated state to inhibit polymerization. GBT021601 has the potential for higher Hb occupancies at lower doses than voxelotor and could potentially reduce treatment burden and improve clinical outcomes. Here we report preliminary safety, efficacy, and pharmacodynamic (PD) data from an ongoing phase 2/3 study of orally administered GBT021601 (NCT05431088) in patients with SCD. Methods: This is a 3-part, multicenter phase 2/3 study in SCD. Part A is a randomized, open-label, 12-week, dose-finding study of GBT021601 in adult participants. Part B is a placebo-controlled study in adult and adolescent participants. Part C is an open-label study in pediatric participants. Here, we report data at Week 12 from Part A, which enrolled participants aged 18-65 years with SCD (HbSS/HbSβ 0 genotype) and Hb 5.5-10.5 g/dL at screening. Eligible participants were randomly assigned 1:1 to GBT021601 100 mg or 150 mg and received a loading dose twice daily for 4 days followed by once-daily maintenance doses through to Week 12. The primary endpoint was change from baseline in Hb at Week 12; secondary endpoints included Hb response (increase from baseline >1 g/dL) and change from baseline in hemolysis markers at Week 12, PK, PD, and safety. Vascular cell adhesion molecule-1 (VCAM-1) levels are elevated during VOCs, with flow adhesion of whole blood on VCAM-1 previously reported as a prognostic biomarker for VOC risk. The effect of GBT021601 on adherent cells was evaluated using a microfluidic device coated with VCAM-1. Results: At data cutoff (June 20, 2023), 35 patients were treated in Part A; 28 had completed 12 weeks of treatment. The 100-mg group consisted of 17/35 patients (48.6%), with a mean (range) age of 29.1 (18-49) years; 7/17 patients (41.2%) were on hydroxyurea (HU). The 150-mg group had 18/35 patients (51.4%), with a mean (range) age of 30.2 (18-59) years; 9/18 patients (50.0%) were on HU. Most patients (32/35 [91.4%]) had the HbSS genotype. After 12 weeks of treatment, the mean (SD) increase in Hb from baseline was 2.67 (1.52) g/dL for the 100-mg group (n=12) and 3.17 (1.82) g/dL for the 150-mg group (n=11). A favorable trend towards reduction from baseline was observed in markers of hemolysis. Flow adhesion data showed patients taking GBT021601 had a reduction in adherent cells, which could correlate with a reduction in the probability of a VOC event (Figure). At data cutoff, the safety data showed GBT021601 was well tolerated. For 27 patients with ≥1 VOC at baseline, the baseline annualized VOC rate was 2.30 (95% CI 1.81-2.92), and the on-study annualized VOC rate was 1.16 (95% CI 0.55-2.43) with a median (range) on-study duration of 0.4 (0.03-0.41) years. Treatment-emergent adverse events (TEAEs), predominantly grade 1 and 2 (15/35 [42.9%]) and unrelated to treatment (14/35 [40.0%]), were reported for 22 patients (62.9%). Treatment-related TEAEs in 8/35 patients (22.9%) were headache (n=4), diarrhea (n=2), and abdominal discomfort, nausea, suspected seizure (uncoded), sickle cell anemia with crisis, upper abdominal pain, and urticaria (n=1 each). There was 1 TEAE, unrelated to treatment, that led to study discontinuation (sepsis, in the 100-mg group). One death deemed unrelated to GBT021601 was due to a cerebrovascular (CVA) event, in a patient with history of CVA and seizures, after new onset high fever and no change from baseline Hb 8.0 g/dL. Conclusions: Loading and daily doses of GBT021601 for 12 weeks were well tolerated in adult participants with SCD. Despite large increases in mean Hb levels, pain episodes did not increase and there was a reduction of adherent cells in a flow adhesion assay with VCAM-1, a potential biomarker for VOC risk. Data from Part A of this phase 2/3 study support the ongoing clinical development of GBT021601 as a potential treatment for individuals with SCD.

Microemboli on Transcranial Doppler in Patients with Sickle Cell Disease: A Single Center Case Series

Background: Stroke remains a major cause of morbidity in patients with sickle cell disease (SCD). Current guidelines recommend that patients with Hemoglobin SS and Hemoglobin S beta zero thalassemia have annual transcranial doppler (TCD) screening and that patients with abnormal velocities should have regular blood transfusions to maintain a maximum HbS level <30% and maintain a hemoglobin level >9 g/dL for at least one year to decrease risk of stroke (DeBaun et al., 2020). While research has shown that patients with velocities >200cm/sec are at increased risk of cerebral infarct, there is no literature to discuss the follow up and management of patients with microemboli on TCD. In the past microemoli were unable to be detected, however TCD is now able to detect such findings (Wan et al., 2022). Studies have been completed on patients with ischemic stroke without SCD and have shown that the presence of microemboli on TCD is associated with a higher rate of recurrence (Das et al., 2020). The presence of emboli has also been shown to recognize patients with asymptomatic carotid stenosis and increased risk for stroke (Markus et al., 2010). Although noted in the general population, the significance of such a finding on an annual TCD screening in patients with sickle cell disease is unknown. Methods:At the Medical University of South Carolina, we performed a retrospective review of all patients with SCD who have had TCDs from 2017 to February 2023 and were found to have microemboli to evaluate management and outcome. Results: We have had 5 patients with an incidental finding of microemboli on their annual TCD since 2017. Three were male with median age of 3 (range 2 to 15 years). Four individuals are HbSS genotype. Mean hemoglobin at the time of TCD was 8.26 ± 1.4 g/dL. TCD velocity was normal in four patients at the time of the TCD, while the remaining individual had a low conditional TCD. Two have a previous history of conditional or abnormal TCDs. The location of the microemboli was variable among patients (Table 1). At the time of microemboli findings on TCD, discussions were held with family regarding options for treatment including chronic partial exchange transfusions and optimization of other disease modifying therapies. Management of all patients included MRI at the time of microemboli, which was normal in only two patients. Three patients were started on chronic manual exchange transfusions for 10 months to 3 years. One out of the 3 continued home HU while receiving transfusions while the remainder were transitioned to HU once chronic transfusions were complete. One patient was not started on chronic transfusions but monitored closely with TCDs due to family choice. On follow up, 4 have had repeat TCDs and all have normalized without any subsequent episodes of microemboli. One patient was found to have silent infarct on MRI at time of TCD and 1 has had follow up MRI 4 years after original TCD with a new silent infarct. Of note, this patient had inconsistent follow up with chronic transfusions after initial abnormal TCD. Importantly, none of our 5 patients had an acute symptomatic ischemic event since the microemboli were noted. Overall, among these patients, 2 have had concern for silent infarct and 3 of 5 started on chronic transfusions (Table 1). Conclusion: This is the first report within the literature of microemboli on annual TCD screening in patients with SCD. As this is a new phenomenon, the significance within the disease course is not known for those with sickle cell disease. Identifying patients with microemboli has been used to help navigate the need for early intervention to prevent stroke. Given that individuals with SCD are already at an increased risk for cerebrovascular events, these findings of microemboli could indicate further risk in these individuals as well and deserve further investigation. Further research is warranted as the significance of such a finding is unknown and further understanding could impact the standard of care of this patient population.

Experiences with Delayed Diagnosis for Older Adults with SCD: “Looking Back It All Makes Sense”

Background Improvements in care over the last 50 years have extended the median lifespan of people living with sickle cell disease (SCD) from 14 years to as long as 61 years in cohorts from academic centers. Older adults with SCD have decades of unique experiences; from navigating their initial SCD diagnosis to coping with complications of SCD as an older adult. The purpose of this study was to describe the impact of delayed diagnosis of SCD on older adults (age ≥ 50 years). Methods We enrolled 19 older adults with SCD from a single comprehensive sickle cell center. We conducted semi-structured interviews by phone or in person. A single interviewer asked open-ended questions about the participant's experiences living with SCD during childhood and adulthood. Participants described their experiences being diagnosed with SCD, how they navigated healthcare systems, and their strategies to manage and prevent pain crises. Interviews were audio-recorded and transcribed verbatim. We used conventional content analysis to code the transcripts. Results The mean age of participants was 58 years (range 50-71). Over half (53%) had HbSS genotype, 37% had HbSC, and 11% had HbSβ+-thalassemia. Almost half (45%) were hospitalized and 63% had an emergency room visit in the last year. Most participants (84%) were on short-acting and 37% were on long-acting opiates. The majority (58%) reported current use of hydroxyurea. In theme 1, many participants described growing up with an unknown illness prior to their SCD diagnosis. Participants discussed having pain episodes but not knowing they had SCD. Their symptoms were often dismissed as “growing pains” or “bone pain”. Participants stated how not having a diagnosis as a child was hard on their parents and they had to see various specialists or travel to academic centers to finally receive a diagnosis. Participants shared stories about how they were diagnosed with SCD (Figure 1). Participants discussed positive aspects of not knowing they had SCD. They described how their parents allowed them to do activities and pursue ambitions without limitations since they were unaware they had a life-limiting condition. In theme 2, participants described the impact of having providers with limited knowledge about SCD. They described challenges having limited access to treatments for pain before and after their diagnosis of SCD. They discussed how doctors were not as empathetic in the past as they are now. Doctors were more likely to stigmatize them as being “drug-seekers”. Some participants stated they still felt “hurt” by how they were stigmatized and remain “very afraid” to seek care in their local hospitals for fear the providers were not knowledgeable about SCD. They recalled difficulties accessing pain medications as children and young adults due to providers' reluctance to treat them. These challenges led them to develop alternative coping strategies aside from opioid medications. Many older adults used therapies such as warm compresses, heating pads, prayer, meditation, healthy eating, proper hydration, and avoiding factors like overexertion and weather extremes. Later in life, these older adults also were more inclined to seek out alternative therapies such as acupuncture, physical therapy, aquatic therapy, and biofeedback to manage their pain. Conclusion Older adults in this study lived for many years not knowing the cause of their symptoms and how best to treat their illness. While delayed diagnosis of SCD is now rare in countries with universal newborn screening, there remains many undiagnosed people with SCD around the world. This delayed diagnosis leads to unnecessary pain and suffering for patients and their families. Participants expressed their frustration with the medical community for their lack of knowledge as to the source of their pain, their dismissive nature of said pain, and their undertreatment of these symptoms. This study highlighted the need for providers to really listen to patients and their families and take their symptoms seriously. There is much to be learned from older adults about self-management of SCD and much work to be done to repair relationships that have been broken due to mistreatment of this population by healthcare providers. In ongoing studies, we partner with older adults with SCD to increase knowledge and develop management strategies, similar to those used in geriatrics, to improve care for older adults with SCD.

Sickle Cell Disease and Hydroxyurea Knowledge, Views and Perceptions Among 400 Sickle Cell Patients and Caregivers at Mulago Hospital Sickle Cell Clinic: A Global Health Study in Kampala, Uganda

Background: In 2017, hydroxyurea was approved for the treatment of all sickle cell patients with genotype HbSS in Uganda following overwhelming support from the NOHARM trial conducted in a malaria endemic region at Mulago National Referral Hospital's Sickle Cell Clinic. Between January 2019 and June 2022, immediately following nationwide approval and distribution of hydroxyurea, hydroxyurea was prescribed a total of 4017 (51.2%) times out of the 7851 HbSS patient visits to the clinic according to data available from the clinic. Aims:The purpose of this study is to investigate the knowledge, perceptions, and beliefs of patients with sickle cell disease and their caregivers attending this clinic in Kampala regarding their disease, complications, genetic impact, general anticipated course of treatment or natural history of disease, and hydroxyurea. Methods: A total of 400 (56%) out of 713 patients were surveyed over a 3-week period using standardized, pretested questionnaires administered orally by trained research personnel in both English (253, 63%) and Luganda (147, 36.8%), which are the most common local languages and dialects spoken by patients in Kampala. Three-hundred and twenty-one (80.3%) participants were caregivers and 79 (19.8%) were children with sickle cell disease between 12- and 17-years-old. Results: Most participants reported receiving counseling about sickle cell disease (333; 83.3%) largely by hematologists (286; 85.9%), however, several knowledge gaps were identified. Two-hundred and ninety-seven (74.3%) participants reported not knowing the type of sickle cell or genotype of the patient. Moreover, presentation of various options did not significantly improve recognition of the correct genotype, with only 121 (30.3%) participants correctly identifying HbSS. Three-hundred and twenty-eight (82%) participants correctly identified the heritable transmission of the disease from both parents; however, caregivers exhibited limited understanding of genetic risk with only 31 (9.7%) correctly identifying the one in four chances of having a subsequent child with sickle cell disease when both parents are carriers. Hydroxyurea counseling was reported by 326 (81.5%) of participants, and 307 (76.7%) participants reported that the patient received hydroxyurea, with most patients taking the medication for at least 1 year. Of the 93 (23.5%) participants who identified the patient had never received hydroxyurea, 32 (34.4%) were uncertain if the patient would be eligible and 56 (57%) believed the patient was eligible to take the medication. Participants identified hydroxyurea benefits as decreases in pain (332; 83%), hospitalization (162; 40.8%), need for blood transfusion (148; 37%), need for pain medication (87; 21.8%), infections (42; 61%), weight loss (22; 31.9%), length of hospital stay (18; 4.5%), and incidence of stroke (10; 2.5%). Slightly more than half of patients had no concerns about hydroxyurea (218; 54.5%). Most patients with concerns (182; 45.5%) identified hydroxyurea's high cost as their main concern (141; 77.5%). Other rumors and fears about hydroxyurea included: negative affects on internal organs such as the liver or kidneys (54; 29.7%), other negative side effects (27; 14.8%), inaccessibility in pharmacies and remote areas (18; 10%), harm due to stopping or missing doses (16; 8.8%), ineffectiveness (16; 8.8%), additional indications as a cancer medication (9; 4.9%), and younger children's difficulty swallowing pills (6; 3.3%). Conclusion:Given the significantly improved morbidity and mortality of sickle cell patients who are adherent to hydroxyurea, it is necessary to address the rumors, misconceptions, and gaps in understanding of the genetic impact of the disease as well as the risks and benefits of hydroxyurea therapy. At the population level, learning is impacted by education levels, literacy rates, health literacy rates, and other cultural and societal factors, thus teaching should be culturally competent and inclusive with verbal and visual methods. The high cost of the medication and lack of consistent supply seem to be the most prohibitive factors in patient acceptance and adherence to take hydroxyurea. With only 1% of Uganda's population covered by health insurance, it is imperative for system-wide changes to address the affordability and accessibility of hydroxyurea and/or to extend the coverage of health insurance to those in need.

Real-World Experience of Individuals with Sickle Cell Disease Treated with Voxelotor: Initial Report from the Multicenter, Prospective Prospect Study

Introduction: Sickle cell disease (SCD) is an inherited blood disorder in which sickle hemoglobin (HbS) polymerization results in red blood cell sickling, which in turn leads to chronic hemolytic anemia, unpredictable pain episodes, and recurrent vaso-occlusive crises (VOCs). Hemolysis and low hemoglobin (Hb) are associated with organ damage, increased morbidity, and early mortality. Voxelotor, a first-in-class HbS polymerization inhibitor, is approved in the US for the treatment of patients with SCD aged ≥4 years and in Europe for the treatment of hemolytic anemia due to SCD in patients aged ≥12 years. In the phase 3, randomized, controlled HOPE (NCT03036813) and the phase 2, open-label HOPE-KIDS 1 (NCT02850406) trials, patients treated with once daily oral voxelotor experienced increased Hb levels and reductions in markers of hemolysis at Week 24 compared with baseline. Here we report the first efficacy and safety data from patients with SCD enrolled in the prospective PROSPECT registry who received voxelotor in a real-world setting. Methods: PROSPECT (NCT04930445) is a post-marketing, open-label, observational, prospective patient registry of patients with SCD (aged ≥4 years) in the US. Eligible participants are being treated with voxelotor as prescribed by their physician as part of their usual care. Participants are treated and evaluated per standard of care and at the physician's discretion. Treatment, including interruptions and restarting treatment, continue at the discretion of the treating physician. Patients receive any additional medications or transfusions as determined and prescribed by their physician. Follow-up duration is 5 years after the first dose of voxelotor therapy. Study data are collected from patient medical records and secondary data sources and entered in case report forms via an electronic data capture system. Results: At data cutoff (May 30, 2023), 85 patients were enrolled at 5 sites. The mean (SD) age was 33.2 (15.3) years, 51.8% of patients were female, and most (92.9%) were Black or African American. Overall, 72.9% of patients were genotype HbSS, 62.4% were taking concurrent hydroxyurea, and mean (SD) Hb at baseline was 7.7 (1.53) g/dL. The mean (SD) duration of treatment, excluding gaps, was 47.0 (12.35) weeks. At the data cutoff, 76 patients (89.4%) continued voxelotor treatment; 7 patients (8.2%) never started voxelotor, and 2 patients (2.4%) discontinued treatment because of adverse events (AEs).The initial prescribed daily doses of voxelotor were 1500 mg (84.7%), 1000 mg (8.2%), 900 mg (4.7%), or 500 mg (2.4%). The most common reasons for prescribing voxelotor were to reduce either anemia (74.1%), frequency of VOCs (15.3%), pain (14.1%), or the frequency of blood transfusion (9.4%); multiple reasons could be selected. In 66 patients with recorded baseline and posttreatment Hb values, the observed mean (SD) peak posttreatment Hb value was 9.0 (1.75) g/dL, representing a mean (SD) increase from baseline of 1.3 (1.25) g/dL. The range of peak Hb change from baseline was −1.4 to 4.6 g/dL and 52/66 patients (78.8%) had a positive Hb response (Figure). Reductions in markers of hemolysis were also observed following voxelotor treatment, with nadir mean (SD) decreases from baseline of −23.9% (55.92%) in reticulocyte percentage (n=54), −1.8 (2.36) mg/dL in total bilirubin (n=61), and −1.6 (1.95) mg/dL in indirect bilirubin (n=38). AEs of special interest reported in >3% of patients included diarrhea, headache, nausea, abdominal pain, and rash (Table); most of these AEs were mild or moderate in severity. Conclusions: PROSPECT is the first prospective patient registry of voxelotor, with the potential to follow up to 1000 participants for up to 5 years and enable long-term, robust data collection of real-world care patterns that could inform how best to incorporate voxelotor into the treatment paradigm of patients with SCD. In this initial cohort of participants enrolled to date, following voxelotor treatment for a mean duration of 47.0 weeks, the mean peak increase from baseline in Hb was 1.3 g/dL, and associated reductions in markers of hemolysis were observed. Safety data were consistent with the phase 3 HOPE and HOPE-KIDS 1 trials. Overall, these initial findings provide support for the real-world safety, tolerability, and effectiveness of voxelotor treatment in individuals with SCD. While enrollment is ongoing, updated data will be regularly reported.

Assessment of Biomarker Response to Clinical Administration of Adakveo in a “Real World” Clinical Setting: A Model for Therapeutic Monitoring in Sickle Cell Disease (SCD)

Background Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) often associated with microvascular blood cell adhesion. P-selectin adhesion has been shown to be a mediator of pathologic adhesive interactions involved in VOEs and is a therapeutic target for SCD-modifying therapies. Adakveo is an FDA-approved (2019) P-selectin monoclonal antibody (mAB) therapy shown to reduce the annualized frequency of VOEs in patients with SCD. Due to the lack of clinically available blood-based biomarkers to define an individual's cellular-level response to therapeutic p-selectin inhibition, providers often rely on the clinical assessment of VOE frequency and intensity to assess an individual's response to anti-p-selectin therapy. This may have contributed to the recently failed Phase 3 clinical trial. In this report, we monitored the cellular-level response to therapeutic p-selectin inhibition by Adakveo administered in a real-world clinical setting using the flow adhesion of whole blood to P-selectin (FA-WB-Psel) assay. Methods This study was a retrospective assessment of FA-WB-Psel biomarker levels before and after clinical administration of Adakveo between January 2019 through June 2022. Flow adhesion of whole blood to p-selectin (FA-WB-Psel ) assays were performed using pulsatile, shear flow (1.67Hz, 1.0 dyne/cm2). SCD patients that received at least 3 biomarker evaluations within 1-year pre-Adakveo and up to 1-year post-Adakveo treatment were included in this analysis. Clinical site received an IRB waiver to confirm therapy start/stop dates. 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 < 0.05 was considered statistically significant. Results There were 15 patients that met the study criteria described above. All patients included were African American with a confirmed HbSS genotype and mean age of 31 years (93% female and 7% male). Of 475 biomarker evaluations, 169 were obtained pre-Adakveo administration, 247 obtained 1-year post-adakveo, and 59 obtained 2-years post-adakveo. Most patients (87%) had FA-WB-Psel biomarker levels above the critical threshold (46 cells/mm 2). Adakveo significantly reduced and stabilized P-selectin activity (FA-WB-PSEL) 12-months (mean pre=84.32 ± 8.44 cells/mm 2, mean post=48.02 ± 4.54 cells/mm2, p=0.001) and up to 24-months (p=0.0003) following Adakveo initiation. Higher pre-Adakveo FA-WB-Psel levels correlate with greater inhibition of cellular-level response to clinically administered Adakveo (r 2=0.4133). Discussion This study provides the first report of FA-WB-Psel adhesion levels in a real-world SCD population initiated on Adakveo therapy. We observed a statistically significant decrease in FA-WB-Psel following Adakveo initiation, suggesting that FA-WB-Psel may be an effective monitoring biomarker for anti-p-selectin therapy in clinical trials and clinical therapy. Patients with low pre-Adakveo FA-WB-Psel levels may be less likely to experience therapeutic p-selectin inhibition, and in fact may be at risk for increased FA-WB-Psel. Studies are underway to assess a potential role for FA-WB-Psel as a predictive biomarker to identify potential Adakveo responders, a monitoring biomarker to serially assess the pharmaco-dynamic response to Adakveo, and a potential surrogate endpoint for therapeutic p-selectin inhibition.

Prevalence of Sleep Disordered Breathing in Adult Sickle Cell Clinic Patients and Cardiovascular Correlates

Sleep disordered breathing (SDB) is strongly correlated with hypertension and cardiovascular disease in the general population, and especially in African Americans. In Sickle Cell Disease (SCD), the adverse effect of nocturnal hypoxemia is anticipated to have additional adverse effects with the sickling of red blood cells and microvascular thrombosis unique to the disease. Prospective data regarding the prevalence and consequences of SDB in this population are lacking. In an ongoing study, 27 adult subjects (age > 18 years), with homozygous HbSS genotype, presenting to the Howard University Sickle Cell Center for routine follow up have been recruited irrespective of the presence of sleep symptoms. Exclusion criteria include previous diagnosis or treatment of a sleep disorder or requirement for supplemental oxygen. Testing occurs when subjects are in a stable phase of disease, with no hospitalizations or medication changes for 4 weeks. In addition to clinical history and questionnaires, participants are assessed with blood work, 6 minute walk testing, pain and sleep diaries during 1 week actigraphy, transthoracic echocardiography and overnight in lab polysomnography with capnography. At the time of this interim analysis, the mean age is 42 (range 29-71, +/- std dev 11) and male:female distribution is 17:10. The mean BMI is 20.4 (+/- 4.5). Mean Hgb is 9.1 g/dl (+/- 1.8) and mean LDH is 325 iu/L (+/- 163). Polysomnography scoring utilized a 3% desaturation criterion for hyponeas as per American Academy of Sleep Medicine Scoring Manual Chapter VIII, 1A. Mean sleep efficiency is 77% ( std dev +/- 4.2%). 48% of subjects demonstrate some degree of sleep disordered breathing as defined by an AHI > 5 events/hour (33% mild, 4% moderate, 11% severe). 70% of subjects demonstrate an RDI > 5 events/hour (40% mild, 19% moderate, 11% severe). Twelve participants (44%) exhibit more than 5 minutes sleep duration at saturations <89% (mean = 187 minutes). Hypercapnia is not suggested as an important component of SDB with the mean ET CO2 = 40.7 mm Hg (range 30-46 mm Hg). Echocardiographic values (LVPWd, Lad, MV E/A, PVV, and RVSP) do not correlate with any indices of sleep disordered breathing other than minutes <89% saturation correlating with RVSP. In summary, this interim analysis of sleep in a population of unselected Sickle Cell Clinic patients demonstrates a striking frequency of sleep disordered breathing. This is despite a generally low BMI for these patients which is typically considered one of the most important risk factor for sleep apnea. Ongoing recruitment is planned to confirm these findings. Correlation with clinical outcomes and potential response to therapeutic interventions will be important future research questions.

Analysis of Volatile Organic Compounds in Exhaled Air in Patients with Sickle Cell Disease during Vaso-Occlusive Episodes

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder characterized by hemolytic anemia, inflammation and vaso-occlusion. Due to the process of vaso-occlusion patients with SCD experience unpredictable and painful vaso-occlusive episodes (VOE), causing stress and disruption of daily life. To date, we lack adequate markers to predict the development of a VOE before it occurs. In other conditions such as asthma, exhaled air analyses have shown to be of diagnostic value and may predict exacerbations. In the human body, metabolic processes generate volatile organic compounds, that are first released into the blood, and then exhaled by the lungs. As a result, exhaled air consists of several volatile, organic compounds that may reflect pathophysiological disease processes. As we presently lack urgently needed biomarkers to predict VOEs, the aim of this study was to identify distinctive volatile organic compounds in exhaled air (eVOC) during VOE in patients with SCD, that may serve as predictors of VOE when detected in low quantities as a VOE is evolving. Methods: In this longitudinal, observational single center cohort study, patients with SCD aged 6 years or older were eligible if they were hospitalized for a VOE at Amsterdam University Medical Centers between October 2021 and March 2023. Following informed consent, an exhaled air sample was taken during hospitalization, and one at least 4 weeks after discharge from the hospital (steady state). Clinical data were collected from medical files including demographics, medical history, medication use and laboratory values during steady state, and during hospitalization for VOE. Patients were instructed not to eat, drink or take medication orally 2 hours prior to the measurements. Exhaled air samples were collected in Nalophan bags, and the eVOCs were transferred to stainless steel thermal desorption tubes filled with Tenax GR for analysis by gas chromatography-mass spectrometry. The Wilcoxon signed-rank test was used to test for differences in eVOCs between VOE and steady state. Significantly different eVOCs were tentatively identified by linking raw chromatograms to corresponding metabolites based on NIST-library matching. The significance level was set to 0.05. All analyses were performed using R Studio (version 1.3.1093) software. Results: In total, 25 patients with SCD were included in this study, providing 71 exhaled air samples. The mean age at inclusion was 25.7 years (SD ±10.2). The majority of the participants had SCD genotype HbSS (60%). At time of exhaled air measurement during hospitalization, participants had symptoms for the median number of 1 day (IQR 0.5-3), and the median pain score was 6 out of 10 (IQR 3-7). A total of 58 different fragments could be identified between VOE and steady state (Wilcoxon rank p< 0.05), representing 26 unique eVOCs. The majority of these eVOCs were alkanes, alkenes, and esters. A representing compound for each chemical group is shown in Figure 1. In the majority of the patients, cyclohexane,1,2,4-trimethyl and propanoic acid, ethyl ester were upregulated during VOE, while 1-propene, 2-methyl- was downregulated. Conclusion: This pilot study identified 26 compounds in exhaled air that differentiated VOE from steady state in patients with SCD. Thus, analysis of exhaled air could serve as a promising, non-invasive and patient-friendly biomarker to predict VOE in SCD. Further identification of the discriminative compounds in exhaled air could provide valuable insights into the metabolic processes during VOE.

National Long-Term Follow-up of Children with Sickle Cell Disease Diagnosed By Newborn Screening in the Netherlands: Overview of the Morbidity and Mortality

Background: Sickle cell disease (SCD) is a genetic red blood cell disorder associated with increased morbidity and mortality. Newborn screening for SCD was introduced in January 2007 in the Netherlands to enable early diagnosis and initiation of preventive measures. The objective of this study was to assess the effect of newborn screening on the morbidity and mortality of children living with SCD in the Netherlands, 16 years after its implementation. Methods: In this national cohort study all children with SCD diagnosed by newborn screening were included. Patients were followed between 1 January 2007 and 31 March 2023 until end of follow-up, death or successful stem cell transplantation. Following informed consent, data were collected from medical files on SCD genotype, treatment, mortality and development of SCD-related complications. The incidence rate of SCD-complications was presented as lifetime-event per 100 person-years for the total duration of follow-up and for the age categories: 0-4, 5-11, and 12-16 years. Results: In total, 391 out of 540 eligible patients (72%) with SCD were included in all hospitals with patients with SCD under treatment, accounting for 3047 person-years. The mean age at the end of follow-up was 7.8 years (SD±4.5). The majority of patients had SCD genotype HbSS (58%), followed by HbSC (28%). As for the mortality, five patients died during follow-up, primarily at or below the age of 2 years (4/5, 80%). In three patients, the cause of death was related to SCD, including infection (n=2; aged 1.2 and 2.0 years) and acute splenic sequestration (n=1; 2.0 years). The SCD-related complications are presented in Table 1. The most common SCD complication in this cohort was vaso-occlusive crisis (including dactylitis) with hospitalization. SCD-related complications that mainly developed within the first 4 years of life, were vaso-occlusive crisis, acute hemolytic crisis, aplastic crisis, severe infections, splenic sequestration and ischemic cerebral infarction. Acute chest syndrome occurred equally in all age categories. Other SCD-related complications developed to a lesser extent in our cohort, and at a later age in adolescence or adulthood. Conclusion: In this cohort of neonatally screened children with SCD, a substantial number of SCD-complications occurred in the first 4 years of life. This study underlines the importance of parental education and 24/7 availably, high level acute specialized clinical care for these children from birth on. Further analysis to identify risk factors at young age for a severe phenotype will be conducted, to enable more targeted interventions in a timely manner. Hopefully, this will lead to reduction of disease burden, improving the quality of life in children living with SCD in the Netherlands.