Pathophysiology Of Sickle Cell Disease Research Articles

Association of Noninvasive Peripheral Blood Hemoglobin Assessments with Venous Blood Draws Among Sickle Cell Patients

Introduction: Sickle cell disease (SCD) is the most common genetic blood disorder, affecting an estimated 100,000 people in the US and over 20 million worldwide. SCD costs one and a half billion dollars of economic loss annually in the US, resulting in sustained burden to the families and the healthcare systems (Value in Health 2021;24:1095). Many patients living with SCD require frequent blood draws and may have to have a central line for routine blood draws. A blood hemoglobin (Hgb) level is the central information about the pathophysiology of SCD including anemia. Low blood Hgb levels have shown as a potential indicator of painful vaso-occlusion crises (Blood 2021;137:2010). Blood Hgb levels are checked for evaluation of new symptoms, medication monitoring, and routine follow up. Real-time and noninvasive monitoring of blood Hgb levels would be clinically beneficial, but less feasible through frequent invasive blood draws. Developing a reliable, self-educated, and completely non-invasive mobile health (mHealth) tool for real time blood Hgb assessments can directly benefit many patients living with SCD, minimizing the number of iatrogenic blood loss from frequent blood draws and improving the quality of care. Methods: Our team has recently developed an mHealth algorithm that can reliably estimate a blood Hgb level from a photo of a peripheral tissue site at individual's inner eyelid (palpebral conjunctiva) (Figure 1a). We conducted a pilot study to determine the exact relationship between peripherally detected blood Hgb levels (accessed from the inner eyelid) and blood Hgb tests (venous blood draws) performed at a clinical laboratory. Fifteen patients with SCD aged from 14 to 73 years were recruited at Hematology Clinic at University Hospital of the Indiana University School of Medicine. Acquisitions of the inner eyelid photos in RAW format were conducted by Samsung Galaxy S21 immediately before or after a blood draw. Blood Hgb levels from the inner eyelid's photo were computed using the previously developed statistical learning algorithm (Optica 2020;7:563); the first step was to recovery a spectrum from an RGB image (spectral learning) and the second step was to compute blood Hgb content using a spectral analysis (Figure 1a). Results: The linear correlation between the peripheral mHealth blood Hgb levels and the venous blood Hgb levels in the blood (i.e., the gold standard) shows a value of 0.82 with a p-value of 0.0002 (Figure 1b). A Bland-Altman analysis was also conducted as a non-parametric statistical method. The limits of agreement (LOA) were defined by a 95% prediction interval of the standard deviation of the differences between the mHealth and conventional blood Hgb levels. 95% Bland-Altman LOA was +/-2.3 g/dL (Figure 1b) and within LOA, the data points were uniformly scattered, indicating no significant bias. This level of performance is comparable to point-of-care blood sampling (finger prick) testing and noninvasive medical devices (Respiratory Care 2019;64:1343). Conclusion: This preliminary study supports the feasibility of peripheral and noninvasive mHealth blood Hgb assessments among patients with SCD. Importantly, peripheral mHealth blood Hgb levels are significantly associated with conventional venous blood Hgb tests among SCD patients. Thus, the mHealth technology can lay the groundwork for empowering individual patients by offering an easily accessible tool for quickly estimating blood Hgb levels by a smartphone. This mHealth technology has potential advantages including mobility, simplicity, and affordability for rapid and real-time blood Hgb quantification among patients living with SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nursing Staff Involvement and Education in Sickle Cell Disease-a Single Institution Quality Improvement Initiative

Introduction: Sickle cell disease (SCD) is the most common inherited blood disorder in the US characterized by vaso-occlusive crisis leading to debilitating pain, organ dysfunction and reduced life expectancy. Despite this, health care staff receive little education on the illness and often feel uncomfortable managing it. The disease is often misunderstood and carries a perceived stigma. Our institution is faced with the additional challenge of lower staff diversity. As part of a larger quality improvement initiative at an institutional level of Sickle Cell Program development, we conducted this project to improve nursing staff level of knowledge and understanding of the disease. Aims: 1) Identify nursing staff who regularly take care of patients with SCD, evaluate their current knowledge and challenges when caring for patients with SCD, 2) Offer educational modules to address knowledge gaps and implement changes to improve comfort in caring for patients with SCD, 3) Provide a pathway that is reproducible for other institutions. Methods: The project was conducted at the University of Wisconsin Hospital by a multi-disciplinary SCD team with collaboration from the Nursing Education specialists and the Diversity Equity and Inclusion department. As a pilot cohort, we identified nursing champions who predominantly take care of patients with SCD and offered them to answer an anonymous survey. Based on their responses, educational modules on SCD were designed and video-recorded. Post-surveys were collected for feedback. The Plan-Do-Study-Act methodology was utilized to document progress and conduct tests of change. QI intervention: Educational module titled "Understanding Sickle Cell Disease and How Nursing Staff Can Help” was designed, video-recorded and uploaded on internal provider platform (U-Learn). Topics included Pathophysiology of SCD, Current treatments, Acute and chronic management of pain, Specialized nursing care for SCD and Addressing disparities, bias and stigma, specifically designed to raise awareness about the new sickle cell team, pain management plans and nursing interventions tailored to our institution. Continuing Education Units (CEU) of 1.5 hours were appointed for their completion. Following the modules, nursing champions answered a post-survey and participated in GRAPES trivia tool, known to improve knowledge and decrease negative attitudes towards patients with SCD. As next step, series of monthly Q&A meetings will be conducted with the nursing champions as an interactive platform for feedback. The nursing champion group will be expanded to form a task force that will serve as SCD patient advocates and help implement ongoing improvements. A best practice alert is being created to facilitate access to patient pain plans in the electronic chart. Patients will be offered surveys to evaluate change in satisfaction with care. Processes implemented at UW will be shared with other regional institutions. Results: Pre-intervention, 15 (65%) staff members completed the survey. The majority requested education on pain management and nursing care (Fig 1). Preliminary findings show that 20 (87%) nursing champions have signed up and 10 (43%) have completed the modules. 30% of members have completed the post-survey. The proportion of respondents reporting good/excellent disease understanding increased to 100% compared to 73% previously (Fig 2). Proportion of staff who report comfort in taking care of patients with SCD increased from 20% to 100% of the respondents (Figure 2). All respondents felt this module provided them with tools to address systematic racism faced by patients with SCD. Pre-intervention, 80% had acknowledged it's presence. Conclusion: Our preliminary data from this QI initiative shed light on deficiencies in the care delivered to an underserved patient population at our hospital, and identified areas of improvement. Lack of knowledge and misconceptions lead to low level of comfort by staff leading to sub-optimal care. Creating a nursing champions cohort interested in improving care of patients with SCD, and empowering them to be part of the change, is a feasible strategy to address institutional and systematic challenges. We started with an interactive CEU based, yet flexible educational strategy to engage staff and using their feedback, introduce ongoing improvements. Similar approach can be adopted at other institutions who face similar challenges. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sickle Cell Disease Compromises the Hematopoietic Stem Cells Bone Marrow Niche with Age

Sickle cell disease (SCD) is characterized by chronic inflammation caused by red blood cell hemolysis, which can trigger oxidative stress and tissue injury that contribute to disease. Recent studies have shown that SCD pathophysiology reconstituted post-transplant in mouse models disrupts the architecture of bone marrow (BM) sinusoidal endothelium and causes a defect of mesenchymal stromal cells (MSCs) function. However, the impact of SCD on the ability MSCs and endothelial cells (ECs) to support of human and mouse hematopoiesis remains largely unknown. Using a mouse model of SCD, we now report that both sinusoidal (LepR+) and arteriolar MSCs (PαS) are more abundant in SCD BM, where they also display increased proliferation at 2-, 6- and 12-months of age. Additionally, 12-month-old SCD mice display a massive loss of sinusoidal ECs. Young SCD patient (n=9, age 2-18) BM also displayed increased MSC numbers relative to age-matched healthy controls (n=13). SCD MSCs isolated from 6- and 12-month old mice, maintained increased cell cycle when cultured ex vivo, showed decreased CFU-F potential, and impaired osteoblast differentiation. In ex vivo co-cultures of primary murine BM MSCs, ECs and HSCs, we found that 2-, 6-, and 12-month old SCD stroma (MSCs and ECs) failed to support non-SCD HSCs relative to controls. Phenotypic HSCs recovered from these co-cultures displayed a loss of CFU potential and transplantation activity. In tri-cultures, SCD MSCs, but not SCD ECs, drove increased HSC proliferation, apoptosis, and lymphoid-biased transplantation. In addition, MSCs isolated from BM of young SCD patients also displayed increased cell cycle, increased pro-inflammatory cytokine secretion, and decreased ability to support the expansion and CFU potential of co-cultured healthy human CD34+ cells, relative to MSCs isolated from young non-SCD donors. Bulk RNA-sequencing of 2-, 6-, and 12-month old murine SCD MSCs revealed gene signatures consistent with accelerated aging, increased proliferation, loss of extracellular matrix (ECM), impaired immune modulation, and reduced secretion of factors known to support HSCs. Genetic restoration of ECM rescued the ability of SCD MSCs to expand HSCs in ex vivo co-cultures, and support HSCs with robust CFU and transplantation potential. In sum, our data reveals a loss of HSPC supportive potential in BM MSCs in young individuals with SCD and that loss of ECM production by MSCs contributes to dysfunction in the HSC BM niche in both young and aged SCD mice.

Dietary Iron Restriction Alters Microbiota-Host Crosstalk and Protects Against Vaso-Occlusion and Organ Damage in Sickle Cell Disease Mice

Introduction: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin, and consequently improved hemolysis and ameliorated anemia. Our lab previously demonstrated that microbial signals from the gut also participated in initiation of VOE and organ damage in SCD mice. To elucidate connections between dietary iron, the microbiota-gut axis, and SCD pathogenesis, we hypothesize that dietary iron restriction may reduce microbiota load and gut permeability, and contributes to amelioration of acute and chronic complications in SCD mice. Methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were conducted 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration in SCD compared to Ctrl group(0.6±0.1 v.s. 4.0±0.7ug iron/mg liver tissue; P=0.001), confirming that dietary iron restriction reverses organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (19.7±0.3 v.s. 20.1±0.2 pg; P=0.006) and serum indirect bilirubin (0.4±0.04 v.s. 0.7±0.07 mg/dL; P=0.001) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice which corresponded with improved VOE parameters (RBC velocity: 2.0±0.2 v.s. 1.6±0.1mm/s; P=0.01; blood flow rate: 314±33 v.s. 230±13pL/s; P=0.01; leukocyte adhesion: 10±1 v.s. 14±1cells/100um vein; P=0.01; IRD v.s. Ctrl). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig1). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig2B). We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds. Microbiota load in IRD treated SCD mice were decreased to 25% of Ctrl level. IRD inhibited translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability. We detected significantly decreased TLR2 ligands in serum of IRD compared to Ctrl mice (0.31±0.04 v.s. 0.46±0.05 TLR2 ligand activities O.D. 650nm; P=0.04) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments under development focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related and novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Efficacy and Safety of L-Glutamine in Preventing Sickle Cell Disease-Related Acute Complications and Hemolysis in Pediatric and Adult Patients: A Real-World Observational Study

Introduction: L-glutamine plays an important role in regulating oxidative stress, one of the key contributors to sickle cell disease pathophysiology. An earlier 48-week Phase 3 study conducted in the United States demonstrated significant reductions in acute complications associated with sickle cell disease, such as vaso-occlusive crises (VOCs) and acute chest syndrome (ACS) in patients on L-glutamine therapy compared to those on placebo (Niihara et al., 2018). This study also showed a significantly fewer number of hospitalizations and hospitalization days for those treated with L-glutamine. Our current real-world observational study is the first to report long term evaluation (120 weeks) of efficacy and safety of L-glutamine (Endari®) therapy in patients living with sickle cell disease in Qatar and French Guiana. Methods: Nineteen patients (10 pediatric and 9 adults), 63% on hydroxyurea, with confirmed diagnosis of sickle cell disease (HbSS genotype) were treated with L-glutamine oral powder (~0.3 g/kg body weight) twice daily for 120 weeks. Of the enrolled patients, 4 patients were from Qatar with the Arab-Indian haplotype and 15 patients were from French Guiana with the African haplotype. The median age was 17 years (range 8 to 54 years) where 53% were <18 years of age. The median body weight of patients was 50 kilograms (range of 25 to 75 kilograms); 53% were males. Clinical events and laboratory parameters (hemoglobin, hematocrit, reticulocytes, and LDH) were collected or measured at baseline, 24, 48, 72, 96, and at 120 weeks. Baseline measures for VOCs, number of hospitalizations, hospitalization days, ACS events, and blood transfusions were collected for the 12 months prior to L-glutamine initiation. Changes from baseline to 120 weeks (annualized) were analysed using MedCalc statistical software Version 20.015. Results: Eighteen patients completed 120 weeks of the real-world L-glutamine treatment observation period. There were significantly fewer VOCs; median of 3 VOCs at baseline and median of 0 at 120 weeks. There were significantly fewer hospitalizations (median of 3 hospitalizations at baseline and 0 at 120 weeks), shorter hospitalization days (median of 15 days at baseline and 0 at end of the observation period), and fewer blood transfusions (median of 3 at baseline and 0 at 120 weeks). The change from baseline values to 120 weeks were significant (p < .00001) for VOCs, hospitalizations, days hospitalized, and the number of units transfused. The number of ACS events were also reduced from 11 at baseline to 0 at 120 weeks. Consistent with these findings, the mean increase in hemoglobin concentrations from baseline was 1.36 g/dL ± 0.17 SE and the mean percent increase in hematocrit from baseline was 5.85% ± 1.10 SE, both significantly improved at 120 weeks (p < .001) Figure 1A. Furthermore, mean reticulocyte counts decreased from baseline (-52.39 x 109/L ± 25.68 SE) and LDH levels decreased from baseline (-311.11 U/L ± 56.02 SE) both significantly changed at 120 weeks (p < .001) Figure 1B. Fewer number of patients were taking hydroxyurea at 120 weeks (8 patients) compared to baseline (12 patients). One patient died during the study (after the 72-week follow up visit) due to multiple deep vein thromboses resulting in a pulmonary embolism; this event was not related to L-glutamine treatment. Conclusion: In this long-term observational study, oral L-glutamine therapy in pediatric and adults living with sickle cell disease resulted in sustained clinical efficacy with improvements in hematologic parameters. L-glutamine therapy was well-tolerated by all patients and there were no safety concerns. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Beneficial effects of a polyherbal formulation in the management of sickle cell disease

Objectives: Inflammation and pain among other comorbid conditions are prominent clinical complications associated with sickle cell disease (SCD). Despite significant improvement in the understanding of SCD pathophysiology, adverse effects of current treatment options are of great concerns. Faradin® (TD) is a polyherbal mixture used in the management of SCD. This study evaluates the acute toxicity, antinociceptive, and anti-inflammatory actions of TD. Materials and Methods: Acute toxicity study of TD was conducted according to test guidelines-423 of the Organization for Economic, Cooperation, and Development. Anti-inflammatory effect was assessed with carrageenan-induced paw edema and xylene-induced ear edema while antinociceptive effect was assessed using tail immersion, acetic acid-induced writhing, and formalin-induced nociceptive methods. Results: Oral administration of TD showed no acute toxic behavior. TD showed non-dose-related inhibition of inflammation in carrageenan- and xylene-induced edema when compared with vehicle-treated control. Post hoc analysis also revealed that TD caused significant increase in pain threshold in acetic acid, formalin, and tail immersion model of nociception. However, TD-induced antinociception was reversed by naloxone (opioid receptor antagonist) indicative of opioidergic system involvement. Conclusion: Findings from this study showed that TD has wide margin of safety and possessed anti-inflammatory as well as antinociceptive properties which lend credence to its potentials in the management of painful and inflammatory conditions associated with SCD.

Impact of Abnormal Leukocyte Count in the Pathophysiology of Sickle Cell Anemia.

Sickle cell disease (SCD) is a hereditary disease that causes deoxygenated erythrocytes to stiffen, resulting in vaso-occlusive crises, endothelial damage, organ failure and systemic consequences. An abnormal leukocyte count is associated with a worse clinical picture in SCD. I studied the link between an abnormal leukocyte count and sickle cell disease. I used the PRISMA guidelines to conduct a systematic review of 5 different clinical literatures that discussed the link. Use of databases including Web of Science, Scopus, Cochrane Library, MEDLINE, ScienceDirect, EMBASE, and JSTOR to make our findings. SCD is a red cell disorder, but an elevated leukocyte count plays a significant role in its pathophysiology. Sickled red cells adhere better to leukocytes when compared to normal red cells, which explains the worse clinical picture seen in sickle cell patients. Hydroxyurea therapy inhibits the recruitment and invasion of neutrophils, which lowers vaso-occlusion. An elevated leukocyte count is responsible for worse clinical manifestations in SCD, eg, clinical stroke, acute chest syndrome, end organ damage, and infarction pain. Leukocytes attach to erythrocytes and vascular endothelium to cause vaso-occlusion, responsible for these effects. They also elaborate inflammatory mediators to aid the process. The higher the leukocyte count, the worse the effects. Hydroxyurea is very helpful in disrupting several pathways of leukocyte contribution to SCD pathophysiology. Therefore, more research on other pharmacologic agents that interfere with this disease process is necessary. Lastly, there is a need for further research into the effects of a reduced leukocyte count in SCD pathophysiology.

Exposing Racial Bias to Confront Care Chasms: Innovations in the Sickle Cell Disease Curriculum at Rush Medical College.

Purpose: Sickle cell disease (SCD) is an autosomal recessive blood disorder that affects 100,000 Americans and 1 in 365 African American births. Individuals with SCD face lifelong complications, such as pain crises, infections, anemia, acute chest syndrome, gallstones, and strokes. Despite the severity and complexity of this condition, it is not uncommon for SCD patients to face significant challenges accessing quality health care. 1 Many providers have implicit biases surrounding treating pain crises with prescription pain medications, and others have not been adequately trained on how to prevent complications or identify changes in organ function. 2,3 Discontinuity of care, disease complexity, and the racialization of “drug seeking behavior” has led to poor outcomes, distrust of the health care system, and a failing therapeutic alliance. Approach: Our vision is to improve SCD patient care by empowering patients, improving care plans, and enhancing medical student education. The latter will be the focus of this abstract, and has been a key focus area in our efforts thus far. We started by formulating a survey to assess Rush students’ baseline knowledge in the domains of racial bias, health inequities, and pathophysiology (pulmonary, neurologic, hematologic, gastrointestinal, and immune systems). After uncovering gaps in knowledge, we began the process of transforming the preclerkship education modules to highlight the complex pathophysiology and psychosocial dynamics of SCD. Patient panels, antibias training, and lectures from specialists are key elements that we are working to incorporate. In addition, we are using Rush resources like the Micro-advocate Simulation Lab to teach empathy and advocacy skills. Outcomes: We gathered data on both preclerkship and clerkship students’ baseline knowledge level to gain insight into the optimal curricular design. Initial survey results of preclerkship students (MS2s, N = 77) in November 2021 showed that only 11.3% of respondents ranked their knowledge level as “knowledgeable/able to teach others about SCD”, with 47.9% of students ranking their knowledge level as “limited/low”. Particular knowledge gaps included medication mechanics, stem cell technology, important clinical labs and inflammatory markers, pathophysiology, screenings for pulmonary and neurologic function, blood transfusion criteria, and the politicization of the Sickle Cell Trait. Clerkship students (MS3s, N = 78) took a similar survey in August 2021, following a focused 2-hour workshop on SCD Pathophysiology & Bias that was piloted this year and featured personal reflections from three patients. Students showed an improvement in every knowledge domain, including qualitative knowledge regarding historic injustice. Of note, 76.8% of clerkship students agreed with the statement, “We need more training on the multiorgan system manifestations of SCD during M1 + M2 year.” Discussion: Our initial results underscore the importance of approaching curricular content with a critical lens that prioritizes equity, advocacy, and patient outcomes. Learners’ SCD knowledge grew significantly after interactive and informative modules that highlighted patient perspectives. Most learners felt these sessions would be best placed in the foundational preclerkship years alongside other “high yield” conditions like Cystic Fibrosis and Hemophilia (which each affect fewer than half the number of U.S. patients than SCD), further underlining the need to empower future generations of physicians to promote social justice. Significance: Our longitudinal project highlights the importance of making sustainable curriculum changes to include diseases that have traditionally been left out despite their prevalence, complexity, and severity. We are currently engaging in a myriad of other SCD initiatives, including planning a health fair in Lawndale, incorporating individualized pain plans into the EMR, designing training modules for different health care roles, and engaging in policy reform with our Illinois House Rep. We are hopeful that our efforts to address SCD-related health inequities will inspire innovative efforts at other hospitals, and we hope to collaborate with other institutions.

PATIENTS WITH SICKLE CELL DISEASE TREATED WITH HIDROXIUREA HAVE HIGHER EXPRESSION OF PD-L1 IN MONOCYTES.

Inflammation plays a key role in pathophysiology of sickle cell disease (SCD). Monocytes are important mediator cells in the inflammation pathway. There is a remarkable lack of knowledge about how monocytes behave in patients with SCD and whether they are modulated by different treatments. To assess possible changes in monocyte maturation and proteinexpression among sickle cell disease patients in different treatment modalities and compare them to healthy subjects. 25 patients with SCD followed at the outpatient service of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and 3 healthy controls were included. We included patients with sickle cell anemia (HbSS), over 18 years of age, without acute complications. Pregnancy was an exclusion criteria. Patients were divided in three groups: treatment with Hydroxyurea (HU) (n = 10) using the same dose for at least three months; chronic exchange transfusion (n = 9) (TP); and no specific treatment (NST) (n = 6). We performed peripheral blood mononuclear cells (PBMC) separation. Monocytes were analyzed by flow cytometry (Dx Flex, Beckman Coulter), using CD14 and CD16 as markers of subpopulations of monocytes as classical (CD14+/CD16-), intermediate (CD14+/CD16+) and non-classical (CD14-/CD16+). We also assessed the expression of CD 86 (a co-stimulation chemokine receptor), CD 163 (receptor for the conjugate hemoglobin-haptoglobin) and PD-L1 (an inhibitor receptor). Comparison between groups were made using ANOVA and Tukey test. Gender and age distribution yielded no significant difference. The mean of total leukocytes and monocytes were, respectively: 8,816 ± 2,133/mm 3 and 900 ± 563/mm 3 (HU), 11,487 ± 3,813/mm 3 and 1,343 ± 754/mm 3 (TP), 9,081 ± 1879/mm 3 and 1,373 ± 432/mm 3 (NST), 7,080 ± 642/mm 3 and 700 ± 0/mm 3 (control).Among the total nucleated cells, the percentage of classical monocytes was lower in HU compared with controls (3.8 ± 2.1%vs.7.7 ± 1.3%, p 0.008), lower in TP vs NST (2.4 ± 1.8%vs. 5.6 ± 2.8%,p 0.04) and in TP vs Controls (2.4 ± 1.8% vs. 7.7 ± 1.3%,p 0.001). No significant differences were shown in the intermediate monocytes. However, nonclassical monocytes were higher in HU compared with TP (0.44 ± 0.3% vs. 0.22 ± 0.2%, p 0.013) and in controls vs TP (0.35 ± 0.07% vs 0.22 ± 0.2%, p 0.017). In classical monocytes, expression of PD-L1 was higher in HU compared with NST (86.1 ± 17.4% vs. 43.9 ± 31.0%) and HU vs Controls (86.1 ± 17.4% vs. 34.9 ± 8.9%, p 0.019). Expression of PD-L1 in intermediate monocytes and in non classical monocytes were also higher in HU compared with controls (88.9 ± 16.3% vs. 58.6 ± 8.6%, p0.02 and 79.6 ± 30.2%vs.37.1 ± 4.6%, p 0.02). Different treatments for sickle cell disease have affected the monocyte subsets distribution and PD-L1 expression in the population studied. HU and Transfusion Program induced a reduction in the Classical Monocyte subset, more pronounced in the latter. The higher expression of PD-L1 in patients submitted to exchange transfusion and in use of HU indicate inhibition of inflammation, which is more pronounced in the HU group. Both treatments have immunomodulatory effects on SCD, however, to date, no inflammatory markers that assess response to treatment are available. Our findings might contribute to uncovering the role of PD-L1 in the chronic inflammation of SCD and to identify a potential biomarker.

The protective effect of the spleen in sickle cell patients. A comparative study between patients with asplenia/hyposplenism and hypersplenism.

Sickle cell disease (SCD) is caused by a point mutation in the beta-globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia, and progressive organ failure. Chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism can also occur with the loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The association of laboratory parameters with the spleen status including hyposplenism, asplenia, and splenomegaly/hypersplenism, and their implication in vaso-occlusive crisis and long-term complications in SCD remain to be determined. We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients: Group a) Patients with asplenia/hyposplenism (N = 22) (including auto-splenectomy and splenectomized patients) vs. Group b) patients with splenomegaly and or hypersplenism (N = 9). Laboratory studies included: Complete Blood Count, reticulocyte count, iron metabolism parameters, C Reactive Protein (CRP), Hb variant distribution, and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release, and K+ leakage, fetal RBC (F-Cells) and F-Reticulocytes, annexinV+, CD71+, oxidative stress measured by GSH presence in RBC and finally Howell Jolly Bodies count were all analyzed by Flow Cytometry. Scanning electron microscopy analysis of RBC was also performed. Patients with asplenia/hyposplenism showed significantly higher WBC, platelet, Hematocrit, hemoglobin S, CRP, D-dimer, Gamma Glutamyl Transferase (GGT), cholesterol, transferrin, annexin V+ RBCs, CD71+ RBCs, together with a markedly lower F Reticulocyte levels in comparison with splenomegaly/hypersplenism patients. In summary, important differences were also found between the groups in the studied RBCs parameters. Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia on laboratory parameters and in clinical manifestations, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach managing hypersplenism in SCD patients should be re-evaluated.

Real-World data on efficacy of L-glutamine in preventing sickle cell disease-related complications in pediatric and adult patients.

BackgroundL-glutamine has been shown to play an important role in the regulation of oxidative stress which is one of the key contributors to the pathophysiology of sickle cell disease (SCD). In a Phase 3 clinical trial, L-glutamine demonstrated a significant reduction in SCD-related complications including vaso-occlusive crises (VOCs), hospitalizations, and acute chest syndrome (ACS) compared to placebo in patients with SCD.ObjectiveThe primary objective was to confirm the efficacy of L-glutamine (Endari®) therapy in pediatric and adult patients with SCD at follow-up time points of 24, 48 and 72 weeks.MethodsIn the observational study, nineteen patients with SCD were treated orally with L-glutamine twice daily for 72 weeks. Clinical and laboratory parameters were measured at baseline and follow-up time points. Patients with severe VOC and ACS were hospitalized. Blood transfusion was given in case of ACS and uncontrolled pain associated with VOC despite administration of the highest dose of intravenous (IV) narcotic.ResultsCompared to baseline, patients had significantly fewer pain crises (median change from 3.0 to 0.0; P < 0.00001), hospitalizations (median change from 3.0 to 0.0; P < 0.00001), days of hospitalization (median change from 15.0 to 0.0; P < 0.00001), and blood transfusions (median change from 3.0 to 0.0; P < 0.00001) at 24, 48, and 72 weeks following L-glutamine therapy. Moreover, there was a drastic decrease in the number of ACS events during this time. A significant increase was observed in mean hemoglobin levels and hematocrit proportions from baseline to 72 weeks (P < 0.001). Conversely, compared to baseline, mean reticulocyte counts and lactate dehydrogenase (LDH) levels were considerably lower at follow-up time points (P = 0.003 and P < 0.001, respectively). No patient reported treatment-related adverse events.ConclusionAlthough the sample size was small, our data clearly demonstrated that L-glutamine therapy was safe and significantly improved clinical outcomes and hemolysis parameters in patients with SCD.

MASP-2 and MASP-3 inhibitors block complement activation, inflammation, and microvascular stasis in a murine model of vaso-occlusion in sickle cell disease

Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients.

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

SummaryIn this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

P1503: REAL WORLD DATA ON EFFICACY OF PHARMACEUTICAL-GRADE L-GLUTAMINE IN PREVENTING SICKLE CELL DISEASE-RELATED ACUTE COMPLICATIONS AND HEMOLYSIS IN PEDIATRIC AND ADULT PATIENTS.

Background: Oxidative stress is one of the key contributors to the pathophysiology of sickle cell disease (SCD) and related complications including acute pain (vaso-occlusive crisis or VOC), tissue ischaemia, stroke, and acute chest syndrome (ACS). L-glutamine (L-gln), a precursor of nicotinamide adenine dinucleotide (NAD), has been shown to play an important role in the regulation of oxidative stress. In the pivotal Phase 3 clinical trial, L-gln demonstrated significant reduction in VOC, hospitalizations, and ACS events compared to placebo in patients with SCD, with or without hydroxyurea (HU) use, over a 48-week period. In September 2021, a re-analysis of the Phase 3 trial data revealed that L-gln decreased the number of VOCs by 45%. Aims: To confirm the efficacy on clinical observations and hemolysis markers of oral therapy with pharmaceutical-grade L-gln in pediatric and adult patients with SCD at follow-up time points of 24, 48 and 72 weeks. Methods: In the observational study conducted from October 2019 through April 2021, 19 patients (4 patients from Qatar and 15 patients from French Guiana) were treated with L-gln (0.3mg/kg) twice daily. Laboratory parameters (hemoglobin (Hb) levels, hematocrit, WBC counts, reticulocyte counts, and LDH levels) were measured at baseline and follow-up time points. Clinical parameters (number of VOCs, hospitalizations, days hospitalized, ACS events, and blood transfusions) were documented for the year prior to treatment initiation as baseline values. These parameters were also collected at 24, 48, and 72 weeks from treatment initiation. Adverse events (AEs) were also collected during the treatment period. The data values at 24, 48, and 72 weeks have been annualized. Statistical analysis was performed using MedCalc 20.015. Results: Compared to baseline, patients had significantly fewer number of VOCs at 24, 48, and 72 weeks following L-gln therapy (median change from 3.0 to 0;p<0.00001). Compared to baseline, there were fewer hospitalizations (median change from 3.0 to 0;p<0.00001) and patients spent fewer days in hospital (median change from 15.0 to 0;p<0.00001). Furthermore, at 24, 48, and 72 weeks, the number of blood transfusions was considerably lower than at baseline (median, from 3.0 to 0.0;p<0.00001). Following treatment with L-gln, the mean Hb level increased significantly from baseline to 72 weeks (8.2 to 8.8 g/dL;p<0.001) with peak mean increase from baseline of 11.2% at 48 weeks. A similar increasing trend was observed for hematocrit proportions from baseline to 72 weeks (24% to 27%;p<0.001) with highest mean improvement from baseline of 15.5% at 48 weeks. Conversely, mean reticulocyte counts and LDH levels were significantly reduced at follow-up time points compared to baseline (p=0.003 and p<0.001, respectively). Image:Summary/Conclusion: This study demonstrated for the first time that L-gln therapy in SCD patients from Qatar and French Guiana resulted in significant improvements in clinical outcomes (number of VOCs, number and duration of hospitalizations, and number of blood transfusions) accompanied by a noteworthy increase in Hb levels and a reduction in markers of hemolysis (reticulocyte counts and LDH levels). Moreover, while 11 ACS events were reported the year before starting L-gln, only 2 ACS events occrred after 12 months of initiation of L-gln. Treatment with L-gln resulted in clinically significant outcomes combined with significant improvement of the hemolysis parameters from baseline through 72 weeks suggesting sustained long-term efficacy. Only a few number of AEs occurred and were mild. No compliance issue was reported.

S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease. Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4. Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events.

S267: PROPERDIN-BLOCKING ANTIBODIES ATTENUATE COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION TRIGGERED BY CELL-FREE HEME IN SICKLE CELL DISEASE MODELS

Background: Complement activation may play an important role in sickle cell disease (SCD) pathophysiology. Heme has been identified as a trigger of complement activation in SCD patients and is present in excess during severe vaso-occlusive crises. Heme is also a DAMP (damage associated molecular pattern) and induces thrombo-inflammation through tissue factor expression. ALXN1820 is a bi-specific VHH nanobody that simultaneously binds human albumin and properdin, thereby effectively and selectively inhibiting alternative pathway (AP) activation and formation of C3 and C5 convertases. The safety of ALXN1820 has been assessed in non-human primates and is currently being tested in a phase 1 study in healthy participants. Aims: 1) To assess if complement activation and heme are functionally linked; 2) To investigate the therapeutic potential of ALXN1820 in in vitro SCD models and in in vivo SCD models using a mouse properdin-blocking monoclonal antibody (mAb), 14E1. Methods:In vitro, complement inactivated C3b (iC3b) and C5b-9 deposition on red blood cells (RBCs) from patients with SCD and complement iC3b and C5b-9 deposition on the endothelial cell line HMEC-1 were assessed by flow cytometry after exposure to heme +/- ALXN1820. In vivo, in Townes SCD mice, vaso-occlusion and hyper-hemolysis were induced by intravenous heme injection (50 µmol/kg) or hypoxia-reoxygenation (8% O2 for 3h then 21% O2 for 1h) in the presence and absence of 14E1 (40 mg/kg), a mouse-specific properdin inhibitor. Vaso-occlusion was measured by staining RBCs with immunofluorescence-labeled Ter-119 antibodies on lung and liver sections. C3b and C5b-9 deposition on SCD RBCs was assessed by flow cytometry. Markers of hemolysis (bilirubin, lactate dehydrogenase, free hemoglobin, free heme) were quantified in plasma using commercial assays. Results: In RBCs from patients with SCD, cell-free heme triggered marked iC3b and C5b-9 deposition (Table). Deposition was blocked in the presence of ALXN1820 by >95% for iC3b and by >85% for C5b-9. HMEC-1 cells exposed to heme showed marked deposition of iC3b and C5b-9. In the presence of ALXN1820, deposition was blocked by >70% for iC3b and >85% for C5b-9. Pretreatment of mice with 14E1 markedly ameliorated vaso-occlusion and reduced both C3b and C5b-9 deposition on RBCs, and hemolysis biomarkers. Image:Summary/Conclusion: These data strengthen the hypothesis that in SCD, cell-free heme is a potent trigger of complement AP activation, which can be blocked by targeting properdin. Investigating the efficacy and safety of anti-properdin ALXN1820 in patients with SCD is warranted as a novel approach to treatment of acute and chronic SCD complications.

PB2222: THE PROTECTIVE EFFECT OF THE SPLEEN IN SICKLE CELL PATIENTS. A COMPARATIVE STUDY BETWEEN PATIENTS WITH ASPLENIA AND HYPERSPLENISM.

Background: Sickle cell disease (SCD) is caused by a point mutation in the beta globin gene. SCD is characterized by chronic hemolytic anemia, vaso-occlusive events leading to tissue ischemia and progressive organ failure. A chronic inflammatory state is part of the pathophysiology of SCD. Patients with SCD have extremely variable phenotypes, from a mild disease to severe complications including early age death. The spleen is commonly injured in SCD. Early splenic dysfunction and progressive spleen atrophy are common. Splenomegaly and hypersplenism, can also occur with loss of the crucial splenic function. Acute, life-threatening spleen-related complications in SCD are well studied. The implications of the spleen status including asplenia and splenomegaly/ hypersplenism in vaso- occlusive crisis and long-term complications in SCD remain to be determined. Aims: In this study, we compared blood parameters and RBC properties of SCD patients and compared the results between patients with asplenia, to patients with splenomegaly/hypersplenism. In addition, we implemented novel, non-routinely used methods for the evaluation of the functional status of the spleen in those patients. Methods: We evaluated the association between the spleen status with clinical and laboratory parameters in 31 SCD patients divided into two groups: Group a) Patients with asplenia (N=22) (including auto-splenectomy and patients that were splenectomized) vs. Group b) patients with Splenomegaly and or Hypersplenism (N=9). Laboratory studies included: CBC, reticulocyte count, iron metabolism parameters, CRP, Hb variant distribution and D-dimer. Metabolic and morphological red blood cell (RBC) studies included: density gradient (by Percoll), glucose consumption, lactate release and K+ leakage; fetal RBC (F-Cells) and F Reticulocytes, annexin V+, CD71+, oxidative stress measured by GSH presence in RBC and finally HJB count were all analyzed by FC. SEM analysis of RBC was also performed. Results: Patients in group (a) showed significantly higher WBC, platelet, HCT, hemoglobin S, CRP, D-dimer, GGT, cholesterol and transferrin levels than patients in group (b). The percent of hyperchromic RBCs, F-Reticulocytes were lower in patients with asplenia. Important differences were also found between the groups in the studied RBCs metabolic parameters. Summary/Conclusion: Further studies are required to elucidate the effect of the spleen including hyper and hypo-splenia in vaso-occlusive crisis, vascular pathology, and long-term complications of SCD. The benefits and risks of splenectomy compared to chronic transfusion need to be evaluated in clinical trials and the standard approach to manage hypersplenism in SCD patients should be re-evaluated.

A phase 1 dose escalation study of the pyruvate kinase activator mitapivat (AG-348) in sickle cell disease

AbstractPolymerization of deoxygenated hemoglobin S underlies the pathophysiology of sickle cell disease (SCD). In activating red blood cell pyruvate kinase and glycolysis, mitapivat (AG-348) increases adenosine triphosphate (ATP) levels and decreases the 2,3-diphosphoglycerate (2,3-DPG) concentration, an upstream precursor in glycolysis. Both changes have therapeutic potential for patients with SCD. Here, we evaluated the safety and tolerability of multiple ascending doses of mitapivat in adults with SCD with no recent blood transfusions or changes in hydroxyurea or l-glutamine therapy. Seventeen subjects were enrolled; 1 subject was withdrawn shortly after starting the study. Sixteen subjects completed 3 ascending dose levels of mitapivat (5, 20, and 50 mg, twice daily [BID]) for 2 weeks each; following a protocol amendment, the dose was escalated to 100 mg BID in 9 subjects. Mitapivat was well tolerated at all dose levels, with the most common treatment-emergent adverse events (AEs) being insomnia, headache, and hypertension. Six serious AEs (SAEs) included 4 vaso-occlusive crises (VOCs), non–VOC-related shoulder pain, and a preexisting pulmonary embolism. Two VOCs occurred during drug taper and were possibly drug related; no other SAEs were drug related. Mean hemoglobin increase at the 50 mg BID dose level was 1.2 g/dL, with 9 of 16 (56.3%) patients achieving a hemoglobin response of a ≥1 g/dL increase compared with baseline. Mean reductions in hemolytic markers and dose-dependent decreases in 2,3-DPG and increases in ATP were also observed. This study provides proof of concept that mitapivat has disease-modifying potential in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

L-glutamine for sickle cell disease: more than reducing redox.

Oxidative stress is a major contributor to the pathophysiology of sickle cell disease (SCD) including hemolysis and vaso-occlusive crisis (VOC). L-glutamine is a conditionally essential amino acid with important roles, including the synthesis of antioxidants, such as reduced glutathione and the cofactors NAD(H) and NADP(H), as well as nitric oxide. Given the increased levels of oxidative stress and lower (NADH):(NAD + + NADH) ratio in sickle erythrocytes that adversely affects the blood rheology compared to normal red blood cells, L-glutamine was investigated for its therapeutic potential to reduce VOC. While L-glutamine was approved by the United States (US) Food and Drug Administration to treat SCD, its impact on the redox environment in sickle erythrocytes is not fully understood. The mechanism through which L-glutamine reduces VOC in SCD is also not clear. In this paper, we will summarize the results of the Phase 3 study that led to the approval of L-glutamine for treating SCD and discuss its assumed mechanisms of action. We will examine the role of L-glutamine in health and propose how the extra-erythrocytic functions of L-glutamine might contribute to its beneficial effects in SCD.Further research into the role of L-glutamine on extra-erythrocyte functions might help the development of an improved formulation with more efficacy.

Editorial introductions.

Editorial introductions.