Homozygous Sickle Cell Disease Patients Research Articles

Electrocardiographic Abnormalities in Adult Homozygous Sickle Cell Disease Patients in Nnamdi Azikiwe University Teaching Hospital, Nnewi, South-Eastern Nigeria

Background; Sickle cell anaemia is the most common monogenic hematologic disorder in Nigeria. Cardiovascular pathology, resulting in electrocardiographic abnormalities are a common finding in adult sickle cell patients.
 Objective; This study investigated the pattern of electrocardiographic (ECG) abnormalities in steady state adult sickle cell anaemia (HbSS) patients and compared the findings with that of age and sex matched Haemoglobin AA(HbAA) controls.
 Methodology; Fifty steady state adult HbSS participants were recruited from the haematology clinic at Nnamdi Azikiwe University Teaching Hospital while fifty age and sex matched HbAA controls were recruited from the hospital community. Participants underwent resting 12 lead ECG using a Schiller CARDIOVIT AT-1 ECG machine. Data was analyzed using Statistical Package for Social Sciences version 21.
 Results; The prevalence of ECG abnormalities amongst the adult sickle cell participants and controls was 70% and 10% respectively. ECG abnormalities were more common amongst male sickle cell participants and left ventricle hypertrophy was the most frequent ECG abnormality detected.
 Conclusion; There is a high prevalence of ECG abnormalities amongst adult sickle cell anaemia patients. Regular Electrocardiography is recommended for early detection of these abnormalities for early treatment and improved quality of life.
 Keywords: Electrocardiographic Abnormalities, Sickle Cell Disease Patients, Nnamdi Azikiwe University, Haemoglobin AA

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

Availability and Cost of Basic Drugs for Sickle Cell Disease in 13 African Countries

Background Sub-Saharan African countries are the hardest hit by sickle cell disease (SCD). Several studies have demonstrated the remarkable efficacy of hydroxyurea (HU) in African sickle-cell populations. As a result, many specialists recommend widespread use of this drug in Africa to reduce the disease's high morbidity/mortality. Nevertheless, in those countries health insurance mechanisms do not exist or and many families affected by SCD do not have enough income to pay for the care. In addition to cost, frequent supply chain disruptions lead to low drugs availability. The cost of basic treatments for SCD and their actual availability in different African countries is not yet known. Yet this information is essential for guiding health policies or the action of non-governmental organizations, and for planning the cost of assistance that could be provided to these patients. The aim of this study was to determine the availability, frequency of supply disruptions and cost of basic treatments for SCDin sub-Saharan African countries. Methods A cross-sectional online international survey was carried out from 24 March to 08 May 2023 among health professionals from French-speaking sub-Saharan African countries following SCD patients and registered in the database of the non-governmental organisation (NGO) Drep-Afrique. Participants completed questions covering the availability (on a scale from 0 (drug not available) to 10 (no availability concern) and the cost of basic drugs in their centres: HU, penicillin V, folic acid, morphine and vaccines. Results 120 health professionals, mainly physicians (91%), from 13 French-speaking Sub-Saharan African countries took part in this study. 58% of the participants worked in SCD referral centres and 36% in university hospitals. 91,000 homozygous SCD patients were reported as being followed in their centres. The median availability score for hydroxyurea and morphine were respectively 5 (IQR 3-8) and 5 (IQR 2-7), compared with 10 (IQR 10-10) for folic acid, 8 (IQR 4-10) for penicillin V, 3 (IQR 1-8) for anti-pneumococcal and 5 (IQR 2-8) for anti-meningococcal vaccines. Frequent supply chain disruptions were declared: 78% for HU, 77% for anti-pneumococcal vaccine, 66% for morphine, 45 % for Penicillin V, while they were rare for folic acid (8%). The median price of HU for one month's treatment of a 30 kg child (mean dose 20 mg/kg/day) was $19.3 (IQR 13.5- 25.3) and varied greatly from country to country (from $38 in Guinea to $10.1 in Togo (p=0.0002) (figure 1). The median price of pneumococcal vaccine was $35.3 (IQR 16.9- 58.7), and thant of meningococcal vaccine $31.4 (IQR 11- 57.5), also with significant differences between countries. The cost of pneumococcal vaccine was 1.6 times higher in centres where the supply chain disrupted than when there is no disruption (p=0.02). Conclusion This study shows that the cost of HU varies from one country to another in French-speaking Africa. Although not very high, it remains unaffordable for the most patients' families. For example, in 2023, the median monthly salary is 238 $ in the Democratic Republic of Congo (the country with the highest prevalence of SCD in the world with Nigeria), compared with a median monthly cost of HU of 25 $ in this country for a child in our study. The high cost of vaccines, beyond the first years of life when a free national program is available in most of African countries, is also a problem. In addition to cost, availability and frequent supply disruptions prevent regular intake of hydroxyurea and, to a lesser extent, of penicillin V. International solidarity and the governments of these countries should work to bring down the cost of hydroxyurea and improve the distribution circuit for these essential medicines.

Hematological indices among young adults with homozygous sickle cell disease in steady state in southwestern Nigeria: a case–control study

Abstract Background: Sickle cell disease is a genetic abnormality involving the haemoglobin synthesis. Although it is a primary red cell disorder, white blood cells (WBC) and platelets are also affected by the mutation. Aim: To assess and compare the hematological parameters of homozygous sickle cell disease in a steady state with age-matched and sex-matched hemoglobin AA controls. Methods: A total of 150 apparently healthy young adults (18–35 years) participated in this study. They were classified into two groups 88 cases with hemoglobin SS and 62 controls. Venous blood collected into EDTA tubes from participants were used for the full blood count analysis which was done using hematological autoanalyzer (SFRI blood cell counter, H18 light, France). The data were analyzed using descriptive and inferential statistics and the alpha value was set at P value less than 0.05. Results: The mean values for white blood cell, mean corpuscular hemoglobin, mean cell volume, red cell distribution width and platelet count were significantly higher in the Hb SS cohort while the packed cell volume and red cell count were significantly lower in the Hb SS cohort. Conclusions: homozygous sickle cell disease patients have lower values of hemoglobin concentration, PCV, the red cell indices but higher values of white cell count and platelets compared with hemoglobin phenotype AA controls.

Fetal Hemoglobin Induced Improvement in Relative Age of Sickle Red Blood Cells Determined through Glycated Hemoglobin Assessment By Flow Cytometry

Introduction: Sickle cell disease (SCD) is characterized by acute vaso-occlusive events and chronic hemolytic anemia, originating from the polymerization of the abnormal hemoglobin S (HbS) under low oxygen tension. Hemolysis is defined as an imbalance between the rate of red blood cell (RBC) production and destruction. It results in an overall reduced lifespan of RBCs, as they are prematurely removed from the circulation. While the normal RBC lifespan is approximatively 120 days, those of SCD is only a sixth of it (Hebbel, 2011). Fetal Hb (HbF) is the main clinical modulator of SCD severity by inhibiting HbS polymerization. Its protective effect relies on its quantity and distribution among the RBC population. However, HbF is often heterogeneously distributed in RBC from SCD patients. Among the numerous improvements triggered by HbF, a significant increase in RBC lifespan has been measured in high-HbF containing cells, as compared to low-HbF containing cells (Franco et al., 2006). As they age, normal RBCs exhibit physiological changes, such as dehydration, a decrease of the intracellular enzymes activities (pyruvate kinase and hexokinase) and an increase in Hb concentration. Glycated Hb (the most abundant being HbA1c form) also increases, as RBC age. The glycation of Hb is a non-enzymatical reaction that is affected by the plasmatic concentration of glucose and the time RBCs last in the circulation. In non-diabetic subjects, the Hb glycation only depends on the RBC lifespan. To date, current methods to assess RBC lifespan are invasive and time consuming, requiring infusion of autologous labeled RBCs and long follow-up. Objectives: This present work aims at 1/ evaluating the RBC relative age of SCD patients using glycated Hb measured by flow cytometry and 2/ estimating the HbF-induced increase in RBC lifespan of SCD patients. Methods: Glycated Hb was measured and compared between five healthy donors (AA), seven non-diabetic and non-transfused homozygous SCD patients (SS) and five non-diabetic, transfused SS patients. Total glycated Hb was measured by reference biochemical methods, cation-exchange high-pressure liquid chromatography or rapid glycohaemoglobin immunoassay (DCA test) used for AA-RBC and SS-RBC, respectively. The single cell glycation was assessed by flow cytometry using mouse anti-human HbA1c antibody. HbA, HbS and HbF were assessed by flow cytometry using anti βA-globin, anti βS-globin and anti g-globin antibodies, respectively. Results: The glycated Hb was significantly lower in SS patients than in healthy donors (glycated HbS =3.7%, glycated HbA =5.3%, p=0.0025 by DCA and HPLC respectively, and by flow cytometry p=0.0061). In transfused SS patients, results showed that glycated Hb was lower in autologous SS-RBCs than in AA-RBCs by a factor of four (P= 0.008), thus confirming the characterized reduced lifespan of SS RBCs. Glycated Hb levels were measured in RBC sub-populations based on their HbF level (high HbF or low HbF subfractions) in non-transfused SS patients. Glycated Hb level was lower in low HbF RBCs, as compared to high HbF RBCs (P=0.0023). This difference was not detected in healthy donors, thereby indicating that HbF has an effect on relative age only in SCD patients. Conclusion: Single cell glycated Hb assessed by flow cytometry allows to simultaneously estimate the relative age of different RBC sub-populations. This new tool is noninvasive and requires only one single time point measurement. It should also be considered to evaluate the efficacy of treatments that aim at improving hemolysis. Figure. Glycated Hb levels assessed by HPLC and DCA biochemical methods, and flow cytometry in different participant groups and RBC subpopulations. Results were expressed as median, percentage or mean fluorescence intensity (MFI) when appropriate. Glycated Hb level was compared between two groups using a nonparametric Mann-Whitney t-test. (A) Total glycated Hb measured by HPLC in AA healthy donors (n=5) and by DCA test in SS patients (n=7). (B) Single cell glycated Hb measured by flow cytometry in SS RBCs (n=7), compared to AA healthy donors (n=4). (C) Single cell glycated Hb measured in low HbF RBCs (n=7) and high HbF RBCs (purple, n=7) subpopulations in SS patients and AA healthy donors (n=4). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P-092: RED BLOOD CELL GLUCOSE CONSUMPTION AND METABOLISM IN SICKLE CELL DISEASE

Purpose: Glucose is the single most essential component of the red blood cells (RBCs), being its sole energy provider and therefore is implicated in the energy metabolomics of the RBCs. This in turn play a crucial role in RBC lifespan, which in Sickle Cell Disease (SCD) patients is reduced to about 20 days from the usual average 120 days. This motivated us to investigate the process of glucose consumption and metabolism in red blood cells of Sickle Cell Patient of Odisha. Materials and methods: Blood samples of homozygous SCD patients (HbSS) were collected randomly with informed consent from different parts of Odisha state depending on the availability and accessibility of the subjects. The samples were studied for their hematological and biochemical parameters including levels of glucose. The red blood cells of SCD patients aged and sex matched normal (HbAA) as well as sickle cell traits (HbAS) were separated and incubated for different time intervals to measure the glucose consumption and Pyruvate and Lactate formation. Along with this the membrane stability of the cells were estimated and time dependent glucose uptake was also carried out. Identification of the haemoglobin variant was done by performing sickling test, haemoglobin electrophoresis and HPLC Hb. Variant analysis. All the biochemical estimations were done following the standard methods. Results: In the red blood cells of SCD cases (HbSS), the levels of glucose consumption were noted to be the higher than the sickle cell trait (HbAS) cases and control/normal (HbAA) samples. Concurrently, formation of blood Pyruvate and Lactate showed higher rate in comparison to HBAA and HbAS red cells indicating raised metabolic turnover in the sickle RBCs. In the time dependent study, the red cells of HbSS patients showed higher rate of glucose consumption compared to trait and normal cells. This data also correlates with the higher hemolytic features of the RBCs showing lower membrane stability with increased metabolism. Conclusion: The sickle cell mutation causes much more damage to the life of RBCs than mere morphological sickling of cells. The energy consumption plays a vital role in the life span, shape, stability and overall biochemistry of the RBCs, thus being a critical factor of RBC metabolism, which in turn determines the overall blood-based health of the SCD patients. Understanding and pin-pointing the key factors of energy metabolism thus holds many potential and promising factors in controlling the sickling, and indeed overall health of patients in case of sickle cell disease. The authors do not declare any conflict of interest

Frequency, pattern, and associations of renal iron accumulation in sickle/β-thalassemia patients.

We evaluated frequency, pattern, and associations of renal iron accumulation in sickle/β-thalassemia. Thirty-three sickle/β-thalassemia patients (36.5 ± 14.7years; 13 females), 14 homozygous sickle cell disease (SCD) patients, and 71 thalassemia major (TM) patients, enrolled in the E-MIOT Network, underwent magnetic resonance imaging. Iron overload (IO) was quantified by the T2* technique. Sickle/β-thalassemia patients had a significantly lower frequency of renal IO (T2* < 31ms) than homozygous SCD patients (9.1% vs. 57.1%; P = 0.001), besides having similar hepatic, cardiac and pancreatic IO. Kidney T2* values were comparable between regularly transfused sickle/β-thalassemia and TM patients but were significantly lower in regularly transfused homozygous SCD patients than in the other two groups. In sickle/β-thalassemia patients, global renal T2* values were not associated with age, gender, splenectomy, and presence of regular transfusions or chelation. No correlation was detected between renal T2* values and serum ferritin levels or iron load in the other organs. Global renal T2* values were not associated with serum creatinine levels but showed a significant inverse correlation with serum lactate dehydrogenase (R = - 0.709; P < 0.0001) and indirect bilirubin (R = - 0.462; P = 0.012). Renal IO is not common in sickle/β-thalassemia patients, with a prevalence significantly lower compared to that of homozygous SCD patients, but with a similar underlying mechanism due to the chronic hemolysis.

A study of left ventricular diastolic dysfunction in patients of sickle cell disease

Background: Left ventricular diastolic filling patterns are altered in patients with sickle cell anaemia and these diastolic abnormalities may be present in the absence of heart failure. These abnormal patterns suggest an intrinsic myocardial abnormality in patients with sickle cell anaemia and may prove to be early markers of cardiac disease. The ventricles do not properly relax and become stiff meaning they cannot fill with blood properly.Methods: This study was carried out in tertiary health care hospital in western India, where homozygous sickle cell disease patients and age and haemoglobin matched controls were taken into cross sectional observational study design.Results: The mean values of E, A, E/A, IVRT, DT, AT, were in normal range in controls. In cases although the mean values of E, A, E/A, IVRT, DT, AT were in normal range, there were 19 cases of sickle cell anaemia who had significant alteration in indices of diastolic LV function from normal range. Out of these 20 cases with diastolic dysfunction, 11 cases had significant increase in (E) velocity from normal range with E/A ratio more than 2 suggestive of restrictive filling pattern of diastolic dysfunction while in 8 cases E value was less than normal with increase in (A) velocity and E/A ratio was less than 1 suggestive of impaired relaxation pattern of diastolic dysfunction. When indices of diastolic LV function were compared in cases and controls, mean early peak filling velocity (E) was significantly higher in cases. Conclusions: In present study, out of 37 cases 19 (51%) cases had LV diastolic dysfunction. Of these 19 cases with diastolic dysfunction, 11 cases had restrictive filling pattern and 8 cases had impaired relaxation pattern of diastolic dysfunction.

Comprehensive Evaluation and Determinant of Second Lactate Threshold in Homozygous Sickle Cell Disease

Following respiratory, cardiac and peripheral vascular impairments, Sickle Cell Disease (SCD) patients can experience early second lactate threshold (S2) during low intensity exercise. Through a comprehensive evaluation of exercise parameters we want to assess and find factors associated with an early reached of S2. Homozygous SCD patients were prospectively included in this study. They performed physical exercise with a standardized protocol. Gaz exchange, capillary lactate concentration measurements and echocardiography were performed at baseline and throughout the exercise until S2 was reached. Baseline and exercise data of the bottom and top half of patients in terms of %predicted VO2 at S2 were compared to investigate the determinant of an early S2, adjusted over age, sex and body mass index (BMI). Thirty patients were included (42 ± 12 years old, 50% of female). Bottom half group reached 34,5% ± 5,2 predicted VO2 at S2 (mean %predicted Watt = 32% ± 5,9) while top half group reached 46,2% ± 6,8 predicted VO2 at S2 (mean %predicted Watt = 42,8% ± 9,6) ( Fig. 1 ). Plasmatic hemoglobin concentration was the only baseline parameter associated with a lower %predicted VO2 at S2 (8.1 ± 1 vs. 9.2 ± 1 g/dl, P = 0.007). None of the baseline echocardiographic parameters were associated with a lower %predicted VO2 at S2. Diastolic adaptation through exercise was associated with a better power and VO2 at S2 (ratio S2/baseline of E’lateral wave and E/E’, respectively 1,39 vs. 1,63, P = 0,039 and 1,2 vs. 0,99, P = 0,004) SCD patients with lower hemoglobin and impaired diastolic adaptation through exercise displayed earlier S2, independently of age, sex and BMI. This highlights the great vulnerability of these patients to low metabolic stress. They should benefit from low intensity physical therapy programs.

Incidence and management of acid peptic disorders in sickle cell disease patients in Western Odisha

Background: Sickle cell disease (SCD) is a chronic hemolytic disorder characterized by the presence of crescent-shaped red blood cells. Abdominal pain is the common presenting symptom in adults with SCD. Among the patients who are not in crisis, it is estimated that one third of patients with homozygous SCD with chronic recurrent epigastric pain has endoscopic evidence of peptic ulcer disease. Aims and Objectives: This study was undertaken with an aim to analyze the incidence and management of acid peptic disorders among SCD patients in Western Odisha. Materials and Methods: A prospective study was done considering 88 homozygous SCD patients. All the patients underwent upper gastrointestinal endoscopy (UGIE) with gastric antral biopsy and rapid urease test (RUT). Cases with Helicobacter pylori were administered a 14 days course of three drug regimen of anti H. pylori drugs and followed-up to 6 weeks of beginning the treatment with repeat UGIE and RUT. Results: Out of 88 cases, 19 cases had duodenal ulcer (21.6%) which was significantly high than patients with gastric ulcer (13.6%). Other findings were gastroesophageal reflux disease and gastritis. About 93.5% of patients became RUT negative after taking anti H. pylori medication. Conclusion: SCD patients with acid peptic disorder constitute a sizeable proportion of patients attending surgical outpatient department and indoor department of surgery. Conservative treatment with life style modification can effectively improve the symptoms of acid peptic disease.

P106: EARLY PREVENTIVE DIAGNOSIS OF HEMOLYTIC ANEMIA IN SICKLE CELL PATIENTS BY DETECTING THE TRIGGERING OF ERYPTOSIS.

Background/Aims: Sickle cell disease (SCD) also known as sickle cell anemia is one of the most worldwide-disseminated hereditary hemoglobinopathies. It is caused by a single amino acid substitution at the sixth residue of β globin gene (Glu6Val), which results in an abnormal hemoglobin called hemoglobin S (HbS). The acceleration of HbS polymerization induces rigid and dysfunctional erythrocytes that play a central role in acute and chronic clinical manifestations of SCD(Conran et al.,2009). Vaso-occlusion and hemolytic anemia are the hallmarks of SCD. We aim to explore the cellular environment of red blood cells to explain the physiopathology of SCD. In fact, the life span of circulating erythrocytes in healthy individuals vary from 100 to 120 days. In SCD, red blood cells undergo a form of cell death, namely, eryptosis before they reach their full life span. Eryptosis is triggered by a wide variety of factors as hyperosmolarity, oxidative stress and energy depletion. It is characterized by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine (PS) exposure (Lang et al.,2012). In this study, we will explore the mechanism of triggering of eryptosis in Sickle cell disease. Methods: Following clinical diagnosis, 50 homozygous SCD patients and 30 healthy donors were identified for hematological and cellular assays. Flow cytometry was performed in order to determine the viability parameters of erythrocytes. The morphology of red blood cells and the externalization of phosphatidylserine was detected by labeling red blood cells with Annexine V. Moreover, we had identified intracellular calcium concentration and ceramide level by labeling erythrocytes with Fluo3-am and anti-ceramide antibodies. Finally, we had quantified reactive oxygen species (ROS) by labeling red blood cells with CM-H2DCFDA. Results and Discussion: Eryptosis in sickle cell patients is accelerated. In fact, PS (+) red blood cells are more present in patients than in healthy subjects. Therefore, eryptosis is triggered by oxidative stress, which stimulates the increase of calcium activity and subsequent externalization of PS and red blood cells shrinkage in sickle cell patients. However, the pathway of ceramide can also be considered a potential stimulating factor of eryptosis in SCD. Eryptosis ensures healthy erythrocyte quantity in circulation, whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in SCD patients.

Role of Antioxidants by Comparative Evaluation of Oxidative Stress in Heterozygous v/s Homozygous Sickle Cell Disease Patients: Research Protocol

Background: Red blood cells are sources for oxidants in Sickle cell anaemia, a genetic disorder of itself. Heme iron and oxygen in oxygenated hemoglobin have bonding interaction and association with electron transfer. In response, antioxidant has modus operandi to reduce oxidative stress and damage to RBC and tissue.&#x0D; The studies on biochemical indicators for oxidative stress in sickle cell would further enhance the understanding and present knowledge of effects of antioxidants on the status of iron metabolism and consequent relief to the sickle cell anaemia patients.&#x0D; Objectives: To evaluate oxidative stress and Antioxidant levels and iron indices factors in heterozygous and homozygous sickle cell disease patients and compare between them along with normal healthy control and Iron Deficiency anaemia&#x0D; Methodology: Comparative Cross-Sectional Study is designed to explore specific antioxidant levels and oxidative stress along with Iron Indices among sickle cell anaemia diagnosed cases attending/admitted in the department of Medicine, Paediatrics and Community Medicine at Jawaharlal Nehru Medical College and its Hospital, Sawangi, Meghe, along with age and sex matched healthy individuals from general population.&#x0D; Results: Oxidative stress as evaluated by stress markers may be higher among the Homozygous sickle cell disease than among the Heterozygous.&#x0D; Conclusion: Oxidative stress may be compounded in sickle cell diseases patients in conjugation with iron deficiency.

Clinical Results of the Drepaglobe Trial for Sickle Cell Disease Patients

▪In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) represents a promising therapeutic option for patients lacking a compatible donor.We previously designed a lentiviral vector (βAS3 LV) expressing a potent anti-sickling βAS3 globin and demonstrated its safety and efficacy in SCD patient cells (Weber et al., 2018). In vitro and in vivo preclinical studies demonstrated the safety and efficacy of a gene therapy (GT) protocol based on the efficient transduction of plerixafor-mobilized SCD HSPCs by βAS3 LV.Based on these results a Phase 1/2 clinical trial (NCT03964792) started in January 2020. Three homozygous SCD patients (P1, P2 and P3) were recruited. Before GT, patients suffered from severe SCD symptoms that were not controlled despite of a strict adherence to the treatment. Plerixafor-mobilized HSPCs were transduced achieving a VCN of 0.9±0.2 in liquid culture, 1.6±0.3 in BFU-E and 0.8±0.2 in CFU-GM. The patients were hypertransfused to reach HbS levels <30%. After busulphan-based myeloablative conditioning, they received a drug product 6 to 8.1x 10 6 per kg.Here, for the 3 patients we report the follow-up of 18 (P1), 10 (P2) and 6 (P3) months. No drug-related severe adverse events were observed. Importantly, no sign of clonal hematopoiesis was detected by NGS sequencing performed on the graft before and after transduction. Despite of the similar VCN in the drug product, gene marking in peripheral blood mononuclear cells was variable and ranged between 0.2 and 0.6, as measured at the last follow-up (0.4 in P1, 0.6 in P2 and 0.2 in P3). These results pinpoint the difficulty to estimate the gene marking, self-renewal and engraftment potential of HSCs in the grafts.P1 and P2 clinically benefit from the GT treatment despite they presented non-severe vaso-occlusive crises (VOCs) not requiring hospitalization (two for P1 at month 6 and 14 and one in P2 at month 8 post-GT). Despite the rapid resolution of these episodes, P1 started hydroxyurea at month 10 and stopped it at month 16. P2 initiated a phlebotomy program to treat liver iron overload and decrease viscosity. Intravascular hemolysis improved in P1 and P2, as shown by a decrease in plasma hemoglobin and heme, and an increase in hemopexin (HPX). Interestingly, in P1 a mild worsening occurred at the time of VOC. In P1 and P2, total bilirubin, a marker of extravascular hemolysis, decreased post-GT. Deformability, measured using osmoscan, improved in P1 and to a lesser extent in P2; however, both still exhibited some degree of dehydration compared to normal RBCs (Fig. 1). Adhesion to thrombospondin decreased after GT with an increase during VOC in P1 or prior the VOC in P2 (Fig. 1). In both patients, markers of inflammation (e.g., C reactive protein) and reticulocyte counts decreased upon GT. An in vitro sickling assays was performed for P1 and showed significant improvement, reaching a frequency of sickling cells similar to SCD heterozygous carriers (from 88% before GT to 45% 6 months post-GT).The GT treatment was not effective in P3 due to the modest intake and rapid decline of gene modified cells.In P1 and P2, who were no longer on transfusions, HbAS3 levels correlated with the VCN (2.1 g/dl for P1 and 3.3 g/dl for P2) and the therapeutic Hb accounted for 21.5%, and 28.7% of the total hemoglobins, respectively. For P3, who remained transfusion-dependent, this frequency was <3% due to the low VCN and the presence of the transfusion-derived Hb. Notably, total Hb concentration was only 8.1 g/dl in P2 before the treatment and reached 11.4 g/dl at the last follow-up, suggesting a better survival of RBCs derived from genetically modified HSPCs. Furthermore, in P1 and P2, HbAS3 levels were significantly higher in mature RBCs compared to reticulocytes, indicating a survival advantage of βAS3 globin-expressing cells.Overall, these clinical data indicate a variable efficacy of the DREPAGLOBE GT treatment, which likely depends on the extent of gene marking achieved in HSCs in vivo and on the engraftment capability of genetically modified HSCs in SCD patients' bone marrow. Single-cell RNA-seq analysis of HSPCs and evaluation of the bone marrow niche in SCD patients will aid to define critical parameters for achieving successful outcomes in GT clinical trials for SCD. [Display omitted] DisclosuresJoseph: bluebird bio: Consultancy. El Nemer: Hemanext: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; AGIOS: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Hemanext: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; Emmaus: Consultancy. Cavazzana: Smart Immune: Other: co-founder.

GBT021601, a Next Generation HbS Polymerization Inhibitor: Results of Safety, Tolerability, Pharmacokinetics and Pharmacodynamics in Adults Living with Sickle Cell Disease and Healthy Volunteers

Background: Sickle cell disease (SCD) is caused by polymerization of sickle hemoglobin (HbS), resulting in red blood cell (RBC) sickling, RBC destruction, vaso-occlusion and end-organ damage. GBT021601 is an oral, small molecule, next-generation HbS polymerization inhibitor. Similar to voxelotor, the first generation HbS polymerization inhibitor, GBT021601 increases hemoglobin-oxygen (Hb-O2) affinity and stabilizes hemoglobin (Hb) in the oxy-hemoglobin (oxyHb) state, thereby inhibiting polymerization of HbS in RBCs. The fraction of Hb bound to drug - (Hb occupancy) approximates the oxyHb molecules per RBC. Compared to voxelotor, GBT021601 has the potential to achieve higher Hb occupancies. GBT021601 achieves greater exposures per dose and is more potent as measured by improvements in hematological parameters in an in vivo SCD mouse model (Dufu, Kobina 2020). We hypothesized that GBT021601 would achieve a substantial reduction in RBC hemolysis and increase in hemoglobin while maintaining a favorable safety profile. We therefore explored safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) in healthy volunteer participants (HVs) and adults living with SCD.Methods: Two studies are in progress: 1) the first in human (FIH), randomized, double-blind, placebo-controlled, parallel group phase 1 study for HVs ages 18-55 years; and 2) a single arm, intrapatient single dose and multiple ascending dose study in homozygous HbSS SCD patients ages 18-60 years with baseline Hb levels ≥5.5 g/dL and ≤10.5 g/dL and without vaso-occlusive crisis or transfusion within 30 days of screening. The HV study tests single ascending doses (SAD) with a 6:2 randomization ratio. Doses administered to HV cohorts ranged from 50 mg to 2200 mg. The SCD patient study tests a single dose (100 mg) followed by multiple ascending doses of the study drug in the same patients to reach a % Hb occupancy of >20-30% over 7 weeks. The primary endpoints for both studies are safety and tolerability. Secondary endpoints include PK and PD. For the patient study, additional secondary endpoints are confirmation of the relationship between time matched GBT021601 concentrations and the change from baseline of clinical measures of anemia and hemolysis. Hb occupancy is calculated as a percentage of the molar ratio of drug concentrations in RBCs to the estimated Hb concentrations (using MCHC) in RBCs.Results: As of July 22, 2021, 63 HVs and 6 adults with SCD were enrolled. Thirty-nine HVs completed the study; one discontinued due to moving to a new geographical area and 23 were in follow-up. GBT021601 was generally well-tolerated, most adverse events (AE) were Grade 1 and 2, there were no deaths, and there was one serious adverse event in a HV that was not related to the study drug. There were no AEs indicative of tissue hypoxia. GBT021601 showed linear PK, high partitioning into RBCs, and a dose dependent increase in percent hemoglobin occupancy in HVs (Figure 1). From single doses of 50 to 1400 mg, the mean preliminary % Hb occupancy ranged from 0.88 to 25%, respectively, exceeding the Hb occupancies reported for HV receiving single doses of voxelotor over a similar range (Putz, 2017).Six adults with SCD have received a single 100 mg dose of GBT021601 and the dose has been well tolerated. Multiple-dose data in adults will be presented.Conclusions: Single doses of GBT021601 are well tolerated in HV and adults with SCD. Given its drug exposure, GBT021601 has the capacity to achieve a targeted Hb occupancy and attain the desired hematological effect at low doses, therefore reducing pill burden and improving clinical outcomes for individuals living with SCD. Forthcoming multiple-dose data will help to evaluate GBT021601's potential as a best-in-class, oral, disease-modifying therapy for SCD.Funding: This study was supported by Global Blood Therapeutics. [Display omitted] DisclosuresBrown: Pfizer: Research Funding; Imara: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Forma Therapeutics: Research Funding; Novo Nordisk: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding. Redfern: Eisai: Other: Advisory Board; Linear Clinical Research: Current Employment; Pfizer: Other: Advisory Board; Novartis: Other: Advisory Board; Astra Zeneca: Other: Advisory Board; Roche: Other: Advisory Board. Lisbon: Global Blood Therapeutics: Current Employment. Washington: Global Blood Therapeutics: Consultancy. Agodoa: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Smith-Whitley: Global Blood Therapeutics: Current Employment.

Association of Clinical and Hematological variables with the disease severity in Indian Sickle cell anemia patients

Sickle cell anemia (SCA) is the most common genetic disorder that is caused due to mutation of the β globin gene. Although SCA is a monogenic disorder, the clinical presentation varies greatly among patients. The present study was designed to be a cross sectional study, aimed at analysing the SCA severity and its association with different clinical, biochemical and hematological variables in SCA patients of Indian origin. About 190 random homozygous SCA patients confirmed by hemoglobin electrophoresis were used in the study. Routine biochemical laboratory (liver function test and Renal function test) and hematologic tests (Total hemoglobin, fetal hemoglobin, hematocrit, MCV and MCH) were done. Values pertaining to complete blood count (CBC), Hb-HPLC and clinical investigations were collected from patient’s records. The mean age of patients with severe disease was significantly lesser than the moderate and mild disease patients. The body mass index (BMI) was also significantly lower in severe disease patients compared to the moderate and mild disease. The patients with severe disease had low levels of red blood cells, total hemoglobin (tHb) and fetal hemoglobin (HbF) compared to the other groups. There is no significant difference in the kidney and liver function among various degrees of disease severity. In summary, this study demonstrates that the tHb and HbF and total leucocyte count (TLC) are major prognostic factors for several clinical complications in SCA. Baseline measurement of these important variables is paramount in predicting important aspects of clinical course and improves the quality lives of these children.

Homozygous sickle cell disease related mortality in Senegal (2011-2020).

Homozygous sickle cell disease (HSCD) is characterized by multiorgan morbidity and an increased risk of early death. We aim to describe the mortality rate, causes, and risk factors of death in HSCD between 2011 and 2020. We conducted a retrospective study with a duration of 10 years in the cohort of 2348 HSCD patients. The mortality rate was determined by reporting the number of deaths to the total number of patients followed in the year. Sociodemographic, clinical, biological data and causes of death were studied. Death risk factors were determined by a bivariate analysis comparing deceased and living HSCD patients. The mean age of death was 26 years (3–52). The sex ratio was 1.2. The mortality rate was 2.76%. The death rate was high in 2011 (3.2%) and low in 2020 (0.17%). We observed a significant reduction of mortality of 94.6%. Most of the common causes of death were acute anemia (40%), acute chest syndrome (24.6%), and infections (20%). Risk factors of death were age, vaso‐occlusive crises ≥3, acute chest syndrome, blood transfusion, and chronic complications. Mortality among HSCD has significantly decreased over the past 10 years in Senegal, and the main causes of death were acute anemia, acute chest syndrome, and infections.

The HBG2 rs7482144 (C > T) Polymorphism is Linked to HbF Levels but not to the Severity of Sickle Cell Anemia.

Sickle cell anemia (SCA) is a severe disease characterized by anemia, acute clinical complications, and a relatively short life span. In this disease, abnormal hemoglobin makes the red blood cells deformed, rigid, and sticky. Fetal hemoglobin (HbF) is one of the key modulators of SCA morbidity and mortality. Interindividual HbF variation is a heritable trait that is controlled by polymorphism in genes linked and unlinked to the hemoglobin β gene (HBB). The genetic polymorphisms that determine HbF levels are known to ameliorate acute clinical events. About 190 well-characterized homozygous SCA patients were included in this study. Complete blood count (CBC), high-performance liquid chromatography (HPLC), and clinical investigations were obtained from patient's records. Severity scores were determined by using the combination of anemia, complications, total leucocyte count, and transfusion scores. HBG2 rs7482144 polymorphism was genotyped by using the polymerase chain reaction and restriction fragment length polymorphism. The association between HBG2 rs7482144 polymorphism and HbF levels as well as the disease severity of SCA were assessed. SCA patients carrying TT genotype were found to have higher HbF levels. In addition, SCA patients with increased severity showed significantly lower levels of hemoglobin, HbF, and hematocrit values. However, the genotypes of HBG2 rs7482144 polymorphism were not found to be associated with the risk of disease severity. In summary, this study demonstrated that HBG2 rs7482144 polymorphism is linked with HbF levels, but it does not affect disease severity. The sample sizes used and the pattern of association deduced from our small sample size prevents us from extrapolating our findings further.

A New Tool for HbF Quantification Demonstrates That Hydroxyurea Decreases Mostly RBCs Containing Very Low Amounts of HbF

Introduction :Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity. Hydroxyurea improves SCD clinical manifestations in part through its ability to induce HbF synthesis which decreases the HbS polymer formation. However, the distribution of HbF is heterogeneous in red blood cells (RBCs) from SCD patients. In the hypothesis of an HbF “threshold” for inhibiting the HbS polymerization, accurate measurement of the HbF content in RBC is an important issue. Several techniques have been developed to quantify HbF. Nevertheless, these technics are qualitative, or semi quantitative, allowing only to detect the RBCs containing a mild/high HbF level, called “F-cells”.Methods : we developed a simple and accurate method to quantify the HbF in each single RBCs and to evaluate the HbF distribution in SS RBCs during a 6 month Hydroxyurea therapy, as a proof of concept. RBCs adults carrying hereditary persistence of HbF (HPFH), β-Thalassemia or db-Thalassemia, were collected. Pancellular homogenous HbF distribution was controlled by flow cytometry. The mean amount of HbF (MCHbF) was calculated from the %HbF determined by HPLC and the mean corpuscular haemoglobin (MCH). The mean fluorescence intensity (MFI) after HbF staining by a monoclonal antibody was quantified by flow cytometry and normalized using quantitation beads.RBCs from homozygous SCD patients were collected before treatment (D0), between 15 days and 3 months (D15-M3) and after 6 months of HU treatment (M6). RBCs were frozen at -80°C. Non-inclusion criteria were vaso-occlusive crisis, blood transfusion within 3 months preceding the enrolment, and on-going pregnancy. After thawing, RBCs were analysed by flow cytometry after HbF staining.Results : Homogenous HbF distribution was proven by flow cytometry in 12 adults with HPFH, δβ thalassemia or β thalassemia. A linear regression between MCHbF and MFI, on 11 different experiments, was performed and provided an equation allowing the quantification of HbF (pg) by RBC (R²=0.94; p<0.0001). A mathematical model calculated the 95% confidence interval. Sixteen homozygous SCD adults were included for the study on HbF distribution under HU. RBCs containing 0 to 2 pg of HbF (table 1) decrease by 25% after 6 months of HU (p<0.0001) whereas a non-significant increase in the other categories of HbF by RBC was found.Conclusion : This simple technique, based on flow cytometry, could represent a new important tool in sickle cell and thalassemia diseases to better quantify the HbF expression and then better evaluate patient's pronostic and therapeutic effects. [Display omitted] DisclosuresGalactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

LRP1 Expression in Sickle Cell Disorders: Insights into the Heterogeneity of Monocytic Response to Heme

Introduction: The importance of heme and its main scavenger hemopexin in the pathophysiology of sickle cell disease (SCD) has been demonstrated in animal models of SCD vaso-occlusion. Increase in monocyte-derived inflammatory cytokines in SCD can be attributed to heme-mediated activation of TLR4 signalling to produce TNF-alpha, and amplification of IL-6 production through a mechanism dependent on heme iron. This suggests that monocytes not only respond to heme but also have receptors to acquire it. We hypothesized that varying levels of heme and hemopexin in SCD could affect how circulating monocytes express LDL receptor related protein (LRP1), the receptor that targets the heme/hemopexin complex to degradation, which can be cleaved to produce a soluble form (s-LRP1).Methods: Peripheral venous blood samples from 119 subjects (39 healthy volunteers - AA, 44 homozygous sickle cell anemia patients - SS, 8 not on hydroxyurea treatment, and 36 hemoglobin SC disease patients - SC) were collected upon signed informed consent approved by the local ethics board. Automated complete blood counts and serum lactate dehydrogenase (LDH), bilirubin levels, and heme and hemopexin concentrations were determined. LRP1 expression was measured by mean fluorescence intensity through conventional flow cytometry (FACS Calibur and FACS DIVA software) in different monocyte subsets, classified according to their surface markers into classical (CD14++ CD16-), intermediate (CD14+ CD16+) and nonclassical (CD14dim CD16+) monocytes.Results and Discussion: As expected, reticulocyte count, LDH, bilirubin, and heme were significantly more elevated in SS than in SC patients when compared to AA subjects, and hemopexin was reduced in both patient groups (P <0.0001). Monocyte subsets differed significantly among the groups: SS and SC patients had more circulating intermediate and nonclassical monocytes than AA subjects (P <0.0001 and P=0.0002, respectively), which is compatible with the systemic inflammation that characterizes SCD. Noticeably, expression of membrane-bound LRP1 was significantly higher in monocytes from SS patients than in those from SC or AA. We also found that LRP1 expression was higher in intermediate and classical monocytes, which are characterized by higher CD14 expression, than in nonclassical CD14dim CD16+ cells. LRP1 expression correlated with hemopexin (rS=0.58, 95%CI 0.26-0.79, P=0.0012) and heme levels (rS=-0.39, 95%CI -0.58 to -0.16, P=0.0012) in SS, but not in SC patients. To address whether monocytic LRP1 expression was associated with circulating LRP1 levels, we measured s-LRP1 and found higher mean s-LRP1 levels in SS than in SC patients (5.52±2.32ug/ml vs. 4.23±2.08ug/mL, P=0.01), but AA subjects showed intermediate s-LRP1 levels (4.70±1.67ug/ml). In SS patients, s-LRP1 correlated with LDH (rS=0.45, 95% CI 0.22-0.64, P=0.0002), but not with all biomarkers of hemolysis, suggesting that s-LRP1 production may also be influenced by mechanisms other than hemolysis. Our data show that more severe hemolysis in SS patients (higher LDH, heme, and lower hemopexin levels) is associated with elevated expression of monocyte LRP1 and its circulating form s-LRP1. Our finding that LRP1 expression in intermediate monocytes correlates with heme and hemopexin levels, particularly in SS patients, suggests this cell type responds to excessive heme. Along with previous publications that this monocyte subtype upregulates heme oxygenase gene HMOX, this supports that monocytes equipped with LRP1 can capture heme/hemopexin complexes and detoxify heme. In addition, higher LRP1 expression in CD14+ circulating monocytes corroborates that these cells can respond simultaneously to free heme with TLR4/CD14-dependent TNF-alpha production and to hemopexin-bound heme captured by LRP1 to foster iron-mediated augmentation of IL-6 production (Dagur et al. 2017).Conclusions: This study highlights that some monocyte subtypes are more prone to capture heme/hemopexin complexes and may react to both free and hemopexin-bound heme, adding to the complexity of the pathophysiology of SCD. Modulation of monocyte differentiation towards subsets with lower responsiveness to heme may be further explored in SCD and other hemolytic diseases in the future.

The NLRP3 Inflammasome in Platelets Is Upregulated in Sickle Cell Disease and Promotes Platelet Aggregation and In Vitro Thrombosis

Sickle cell disease (SCD) is a severe monogenic disorder characterized by chronic hemolysis and abnormal coagulation and inflammation. Activated platelets play a critical role at the interphase of thrombosis and inflammation in the disease context. Sensing of free heme, a degradation byproduct of hemoglobin oxidation during hemolysis, involves the pattern recognition receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3), which typically forms multiprotein inflammasome complexes and controls caspase-1 activity and cleavage of IL-1β not only in immune cells but also platelets. We have shown recently that the NLRP3 inflammasome in platelets controls platelet activation, aggregation, and in vitro thrombus formation, which was dependent on platelet bruton's tyrosine kinase (BTK). The role of the platelet NLRP3 inflammasome and BTK in SCD, however, is unexplored.Platelets were isolated from humanized SCD mice (Townes), which express human sickle hemoglobin, and patients with SCD in steady state and acute vasoocclusive crisis. Platelet NLRP3 activity, as monitored by caspase 1 activation (assessed by fluorescent labeled inhibitor of caspase-1, FLICA), and cleavage and secretion of IL1β (assessed by Western blot and ELISA), was significantly elevated in SCD patients in steady state as compared to healthy subjects (FLICA: mean 340.3 ± 30.2 SD vs. mean 262.2 ± 33.9 SD, p<0.001), and further upregulated in SCD patients in crisis (FLICA: mean 390.3 ± 34.0 SD vs. mean 340.3 ± 30.2 SD, p<0.05). NLRP3 activity in platelets was also significantly increased in homozygous SCD mice, as compared to heterozygous and control mice (FLICA: mean 165.1 ± 32.4 SD vs. mean 86.7 ± 20.5 SD, p<0.001), which was significantly reversed by the heme scavenger hemopexin or pharmacological inhibition of the NLRP3 inflammasome/BTK pathway. Moreover, incubation of platelets with heme induced intraplatelet colocalization of NLRP3 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), as evaluated with immunofluorescence staining coupled with confocal laser scanning microscopy, indicating the assembly of the NLRP3 inflammasome in platelets upon exposure to heme. We studied the functional effects of platelet NLRP3 inflammasome upregulation in SCD, and found that collagen-induced platelet aggregation was significantly elevated in homozygous SCD mice as compared to control animals (area under the curve, AUC: mean 65.25 ± 7.99 SD vs. mean 40.60 ± 7.92 SD, p<0.05). In SCD patients in acute crisis, aggregation of circulating platelets was significantly upregulated as compared to patients in steady state (AUC: mean 113.70 ± 16.26 SD vs. mean 78.53 ± 1.93 SD, p<0.05). Moreover, platelet aggregation in steady state patients was significantly elevated as compared to healthy subjects (AUC: 78.53 ± 1.93 SD vs. mean 30.87 ± 4.34 SD, p<0.01). The upregulated platelet aggregation responses in homozygous SCD mice and SCD patients in acute crisis were dependent on heme/NLRP3/BTK pathway, as evaluated by hemopexin (mice: 69.3 ± 5.7% inhibition; patients: 61.6 ± 5.2% inhibition), the NLRP3 inhibitor MCC950 (mice: 75.7 ± 3.9% inhibition; patients: 72.1 ± 3.8% inhibition), and the BTK inhibitor CGI1746 (mice: 65.9 ± 3.6% inhibition; patients: 67.1 ± 4.1% inhibition) (p<0.01). Similar results were obtained with a collagen-coated flow chamber system.Our data show that the platelet NLRP3 inflammasome is upregulated in SCD via heme, which results in elevated platelet aggregation and in vitro thombus formation, and is potentially targetable using BTK inhibitors for affected patients. DisclosuresNo relevant conflicts of interest to declare.