Steady-state Sickle Cell Disease Research Articles

Ultrasonographic assessment of spleen size and pattern of change among sickle cell disease patients and healthy controls in North-Eastern Nigeria

Background: Ultrasonography is an established and reliable method for assessing the spleen. Because of variation due to genetic and other environmental factors including malaria endemicity, interpretation of spleen sizes requires a knowledge of the normal reference range for a given population. This study aimed to identify spleen size reference ranges across age groups of healthy controls to serve as a baseline to assess changes in spleen size in patients with sickle cell disease. Methods: Using a cross-sectional study design, spleen size was measured in healthy people of different age groups and steady-state sickle cell disease patients (children and adults) using abdominal ultrasonography. Using the age-group-specific reference values obtained from the controls, spleens were classified into small, normal size or enlarged among the sickle cell disease patients. Results: The study consisted of 109 (34.8%) healthy controls and 204 (65.2%) steady-state sickle cell disease patients. The spleen was visualised in all the controls ( n = 109) and in 107 (52.4%) sickle cell disease patients. Using cut-off values for spleen length among the controls across age groups (< 5 years (5.0–7.0 cm); 5–9 years (5.5–8.5 cm); 10–14 years (6.0–11.0 cm) and ⩾ 15 years (7.0–12.5 cm)), spleen size was classified as small ( n = 18/204; 8.87%), normal ( n = 68/204; 33.3%) and enlarged ( n = 21/204; 10.3%) among the sickle cell disease patients. Conclusion: Model-based age-group reference ranges and percentile curves for splenic dimensions based on ultrasonography among normal controls in North-Eastern Nigeria were established and may be of value in assessing spleen sizes among sickle cell disease patients living in malaria-endemic regions of Africa.

Influence of hydroxyurea on tubular phosphate handling in sickle cell nephropathy

ObjectiveThis study aims to evaluate the markers of tubular phosphate handling in adults with sickle cell anemia (SCA) and the influence of hydroxyurea (HU), the degree of anemia and Hb F concentration on these markers. MethodsEighty-eight steady state SCA patients in outpatient follow-up in Fortaleza, Ceara, Brazil and 31 healthy individuals were included in this study. Vitamin D (25OHD) was measured by enzyme-bound fluorescence assay, intact parathyroid hormone (iPTH) by electrochemiluminescence, and serum and urinary phosphate and creatinine by colorimetric methods. Details of Hb F and HU use were obtained from clinical records. Tubular reabsorption of phosphate (TRP) and maximum tubular reabsorption of phosphate (MTRP) were calculated. SCA patients were stratified according to the use of HU, degree of anemia and percentage of Hb F. The significance level was set for p-values <0.05. ResultsCompared to controls the 25OHD level (25 ± 11 vs. 30 ± 9 pg/mL) was lower in SCA, while serum phosphate and MTRP were higher (3.86 ± 0.94 vs. 3.46 ± 0.72 and 3.6 ± 1.21 vs. 3.21 ± 0.53, respectively). There was no significant difference in iPTH, TRP and phosphaturia. Serum phosphate showed correlation with TRP (r = 0.32; p-value = 0.008) and MTRP (r = 0.9; p-value <0.001) in SCA. Patients taking HU, especially those with Hb F >10 % presented reduced serum phosphate levels, and TRP and MTRP rates. Those with mild anemia presented reduced serum phosphate levels and MTRP rates. ConclusionSerum phosphate levels and renal phosphate reabsorption rate were increased in SCA. HU use, high Hb F concentration and total Hb were associated with better control of tubular phosphate handling markers.

OBSERVATIONAL STUDY ON SILENT CEREBRAL INFARCT IN CHILDREN OF SICKLE CELL DISEASE IN ASSOCIATION WITH RISK FACTORS

Aim and Objectives: To find out the presence of silent cerebral infracts (SCI) among sickle cell diseased children and to identify risk factors and correlate risk factors with silent cerebral infracts in sickle cell diseased children.. Settings, Design. Methods and Materials: The children who came to paediatrics department of maharaja institute of medical sciences, with confirmed haemoglobin electrophoresis findings of sickle cell disease in steady-state are subjected to T2 weighted MRI scan of the brain using Philips ingenia CX 1.5Tesla MRI machine. Results: out of 50 children with sickle cell disease ,18(36%) children have silent cerebral infarct and it was observed that the lower hemoglobin concentration, Lower RBC count, Lower Serum Ferritin, and elevated WBC count were considered to be the potential risk factors for Silent cerebral infarcts in Sickle cell disease. Conclusion : Patients with risk factors for silent cerebral infarcts should be evaluated for cerebrovascular disease. If evidence of the presence of SCI, consideration must be given to therapeutic intervention.

Malaria Infection in Patients with Sickle Cell Disease in Nigeria: Association with Markers of Hyposplenism.

Malaria is considered an important cause of morbidity and mortality among people living with sickle cell disease (SCD). This has partly been attributed to the loss of splenic function that occurs early in the disease process. We conducted a cross-sectional study and determined the frequency of malaria infection among SCD patients and explored the association with spleen's presence on ultrasonography and spleen function assessed using the frequency of Howell-Jolly bodies (HJBs). A total of 395 participants consisting of 119 acutely-ill SCD patients, 168 steady-state SCD controls, and 108 healthy non-SCD controls were studied. The prevalence of Plasmodium falciparum parasitemia was 51.3% in acutely-ill SCD patients, 31.7% in steady-state SCD controls, and 11.0% in the healthy non-SCD controls; however, the mean parasite density was significantly higher in the non-SCD controls compared to both SCD groups (p = 0.0001). Among the acutely-ill SCD patients, the prevalence of clinical malaria and severe malaria anemia were highest in children <5 years of age. The prevalence of parasitemia (p = 0.540) and parasite density (p = 0.975) showed no association with spleen presence or absence on ultrasonography. Similarly, the frequency of HJB red cells was not associated with the presence of parasitemia (p = 0.183). Our study highlights the frequency and role of malaria infection in acutely-ill SCD patients, especially in those younger than five years. Although we have found no evidence of an increased risk of malaria parasitemia or parasite density with markers of hyposplenism, the role played by an underlying immunity to malaria among SCD patients in malaria-endemic region is not clear and needs further studies.

Prevalence and pattern of sensorineural hearing loss among children and adolescents with sickle cell disease in a tertiary health facility, Northwest Nigeria

Sickle Cell Disease (SCD) can result in painful vascular occlusion crises, anoxia, and ischemia, which can occasionally cause damage to tissues and organs, including the auditory system, particularly the blood-rich cochlea. Despite being underreported, Sensorineural Hearing Loss (SNHL) is a well-known consequence of SCD globally. The study’s objective was to determine the prevalence and pattern of hearing loss in children and adolescents aged 5 to 16 years old with SCD who were in steady state and were seen at the hematology and pediatric outpatient clinics at Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria. A prospective hospital-based case-control study was conducted with 125 children with SCD in steady state, aged 5 to 16, who were enrolled in the hematology clinic at the Ahmadu Bello University Teaching Hospital in Zaria, Nigeria, and 125 ‘age- and sex-matched healthy controls, who were selected from the ABUTH’s pediatric outpatient clinics. An organized questionnaire was used to collect fundamental data on the sociodemographic characteristics and medical history of the participants and controls. In a sound-treated booth, the participant’s hearing was evaluated using tympanometry and pure tone audiometry. Overall, 68 (54.4%) and 57 (45.6%) out of the 125 assessed participants were male and female, respectively, with a male-to-female ratio of 1.2:1 and the mean age 10.17±3.55 years. Due to age-sex matching, the subject’s age and sex distributions match those of the controls. Children with SCD experienced bilateral SNHL ≥25 dB in 32/125 (25.6%) cases (21 males;11 females). The control group, which had a HbAA phenotype that was normal, did not exhibit any hearing loss. 21/32 (65.6%) of the participants had mild (26-40 dB) hearing loss, and SNHL occurred more frequently in males (21/32 (65.6%) than in females (11/32 (34.4%). In comparison to 3(9.4%) HbSC and 1(3.1%) HbSS+F, SNHL was more common in individuals with 28(87.5%) HbSS phenotypes. While diverse frequencies were impacted in the affected participants, there was no consistency in the frequency pattern of hearing loss. The current study showed that SNHL is a frequent complication in children and adolescents with SCD. About 25% of children and adolescents with SCD experienced SNHL, which disproportionately affected males. Frequent audiometry should be carried out to check the children’s hearing levels and identify any early hearing losses so that interventions can be made to perhaps prevent associated speech and language issues that might cause educational challenges.

Isoquercetin for thromboinflammation in sickle cell disease: a randomized double-blind placebo-controlled trial

AbstractData from a small trial in patients with cancer suggest that isoquercetin (IQ) treatment lowered thrombosis biomarkers and prevented clinical thrombosis, but, to our knowledge, no studies of IQ have been conducted to target thromboinflammation in adults with sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled trial in adults with steady-state SCD (hemoglobin SS [HbSS], HbSβ0thal, HbSβ+thal, or HbSC). The primary outcome was the change in plasma soluble P-selectin (sP-selectin) after treatment compared with baseline, analyzed in the intention-to-treat population. Between November 2019 and July 2022, 46 patients (aged 40 ± 11 years, 56% female, 75% under hydroxyurea treatment) were randomized to receive IQ (n = 23) or placebo (n = 23). IQ was well tolerated and all the adverse events (AEs; n = 21) or serious AEs (n = 14) recorded were not attributable to the study drug. The mean posttreatment change for sP-selectin showed no significant difference between the treatment groups (IQ, 0.10 ± 6.53 vs placebo, 0.74 ± 4.54; P = .64). In patients treated with IQ, whole-blood coagulation (P = .03) and collagen-induced platelet aggregation (P = .03) were significantly reduced from the baseline. Inducible mononuclear cell tissue factor gene expression and plasma protein disulfide isomerase reductase activity were also significantly inhibited (P = .003 and P = .02, respectively). Short-term fixed-dose IQ in patients with SCD was safe with no off-target bleeding and was associated with changes from the baseline in the appropriate direction for several biomarkers of thromboinflammation. The trial was registered at www.clinicaltrials.gov as #NCT04514510.

The impact of interferon-gamma level on the health status of patients with sickle cell disease in Basrah

Sickle cell disease (SCD) is one of the most prevalent autosomal recessive diseases, characterized by the generation of abnormal hemoglobin S. Our study aimed to assess how the serum level of interferon-gamma affects the health status of patients with SCD in Basrah. A total of 90 participants were enrolled in this study and divided into two main groups: a SCD group and a control group. The SCD group included 30 patients with SCD in steady state and 30 patients with SCD in vasoocclusive crisis; the control group included 30 ageand sexmatched apparently healthy individuals. Approval was obtained from the Research Ethics Committee of the College of Medicine, University of Basrah before conducting the study. Two milliliters of venous blood were drawn from all the participants, and ELISA tests were utilized to determine the levels of serum interferon-gamma. There was a statistically significant increase in the serum level of interferon-gamma among SCD patients (both in steady state and in crisis) compared to the control group (p = 0.05). There were no significant differences in the levels of interferon-gamma between the patients in steady state and during vaso-occlusive crisis (p &gt; 0.05). Interferon-gamma may influence the general health of sickle cell patients and contribute to the cause of inflammation, no matter whether the patient is in stable condition or is experiencing a crisis.

Impact of splenectomy on circulating microparticles in patients with sickle cell anemia.

Circulating microparticles (MP) are being described as potential biomarkers for disease activity in a variety of conditions including sickle cell anemia (SCA). However, relatively little is known about the influence of spleen status on MP levels in patients with SCA. Using a prospective study design we characterize circulating MP in 144 patients with SCA in steady state by assessing their cellular origin and their relationships to spleen status defined by clinical and imaging findings. In addition, MP levels were studied according to demographic characteristics, clinical status, treatment modalities, and other hematological and biochemical parameters. Absolute plasma concentrations of MP were determined by flow cytometry. Patients with SCA displayed a 10-fold increase in levels of MP derived from red blood cell (RBC) and platelets (PLT) when compared to their healthy counterparts (p < 0.0001). Splenectomized patients with SCA have more pronounced levels of MPRBC and MPPLT, and remained elevated after several weeks of follow-up. Levels of MP were not significantly associated with spleen removal procedures, age, gender, clinical severity score, hydroxyurea therapy, hemoglobin F, and co-existence of glucose-6-phosphate dehydrogenase deficiency. Collectively, these results suggest that splenectomy affects circulating levels of MP regardless of the known SCA modifiers and correlates.

#4385 RELEVANCE OF REPEATED ALBUMINURIA AND GLOMERULAR FILTRATION RATE IN A LARGE COHORT OF SICKLE CELL ANEMIA CHILDREN LIVING IN A RESOURCE-LIMITED AREA

Abstract Background and Aims Chronic kidney disease (CKD) is associated with significant morbidity and mortality among patients with sickle cell anemia (SCA). Glomerular hyperfiltration (GHF) and albuminuria are known as early manifestations of kidney disease occurring in early childhood and can predict the progression to CKD in these patients. Studies reported the prevalence of these kidney abnormalities in SCA children using a single measure and their association with genetic risk factors, especially the co-inheritance of APOL1 risk variants (RVs). However, data on the prevalence of persistent kidney abnormalities (based on the KDIGO CKD definition) and their association with APOL1 RVs are limited in SCA children, particularly those living in sub-Saharan Africa. This study aimed: (i) to determine the prevalence of persistent kidney abnormalities (albuminuria and/or GHF) in SCA children living in the Democratic Republic of Congo (DRC); and (ii) to assess the association between persistent kidney abnormalities with clinical and genetic risk factors. Method From March 2021 to December 2022, we prospectively enrolled 585 steady state SCA children aged 2 to 18 years (male gender 278/585; 47.5%). The SCA status was confirmed through the molecular sequencing of beta-globin gene. Clinical and biological parameters were obtained. All participants were genotyped for apolipoprotein-L1 (APOL1) G1 (rs73885319, rs60910145) and G2 (rs71785313) variants. APOL1 high-risk genotype (HRG) was defined by the presence of 2 risk variants (G1/G1, G2/G2, and G1/G2), and low-risk genotype (LRG) by the presence of 0 or 1 risk variant. Albuminuria was defined as urinary albumin-to-creatinine ratio (ACR) ≥ 30mg/g. The estimated glomerular filtration rate (eGFRcr) was calculated using the original Schwartz formula and GHF was defined as eGFRcr ≥ 180 ml/min/1.73 m2 for children between 2-10 years of age and &amp;gt; 140 ml/min/1.73 m2 for children more than 10 years of age. All measurements were repeated at least three months later in participants who presented with kidney abnormalities at the first screening. The main outcome parameter was persistent albuminuria or persistent GHF for more than three months. Results At enrollment, 234/585 (40.0%) participants presented with kidney abnormalities, among which 80/585 (13.7%) with albuminuria and 171/585 (29.2%) with hyperfiltration. From participants found with kidney abnormalities at the first screening, 176/234 (75.2%) were available for repeated screening: 56/176 (31.8%) presented with persistent kidney abnormalities and 120/176 (68.2%) had a regression of kidney abnormalities without any treatment. Out of 176 participants who benefited from repeated screening, 57 had baseline albuminuria and 130 had baseline GHF. Persistent albuminuria and persistent GHF were found in 38.6% (22/57) and 28.5% (37/130), respectively. Multivariate logistic regression revealed that APOL1 HRG was significantly associated with persistent albuminuria (OR 3.4, 95CI 1.1-10.7, p = 0.037), while male gender was significantly associated with persistent GHF (OR 2.1, 95CI 1.0-4.2, p = 0.042). Conclusion A significant proportion (almost 70.0%) of SCA children who had kidney abnormalities at the first measure, presented regression of these abnormalities without any reno-protective drugs. This result emphasizes the importance of repeating screening at least three months later to confirm CKD, as recommended by the KDIGO Guidelines. This strategy will reduce the need for unnecessary treatment, which represents a high financial burden in a resource-limited area.

Levels of inflammatory markers are differentially expressed in sickle cell anemia and sickle cell trait.

Although sickle cell anemia (SCA) is related to inflammation, the profile of inflammatory markers in sickle cell trait (SCT) is poorly studied. This is a cross-sectional study of inflammatory biomarkers carried out involving adults with SCA in steady state, SCT and controls. The SCA group had higher levels of lactato dehydrogenase, IL-1β, IL-6, IL-10, and tumor necrosis factor alpha than the others, while the SCT group had similar levels to control group. In addition, SCA group had lower IL-8/IL-10 and soluble triggering receptor expressed on myeloid cells-1/IL-10 ratios. These findings indicate that individuals with SCT do not have a chronic inflammatory profile and reinforce that cytokines are involved in the maintenance of the inflammatory state in SCA.

Evaluation of two red cell inclusion staining methods for assessing spleen function among sickle cell disease patients in North-East Nigeria.

The loss of splenic function is associated with an increased risk of infection in sickle cell disease (SCD); however, spleen function is rarely documented among SCD patients in Africa, due partly to the non-availability of sophisticated techniques such as scintigraphy. Methods of assessing splenic function which may be achievable in resource-poor settings include counting red blood cells (RBC) containing Howell Jolly Bodies (HJB) and RBC containing silver-staining (argyrophilic) inclusions (AI) using a light microscope. We evaluated the presence of HJB-and AI-containing RBC as markers of splenic dysfunction among SCD patients in Nigeria. We prospectively enrolled children and adults with SCD in steady state attending outpatient clinics at a tertiary hospital in North-East Nigeria. The percentages of HJB-and AI-containing red cells were estimated from peripheral blood smears and compared to normal controls. There were 182 SCD patients and 102 healthy controls. Both AI- and HJB-containing red cells could be easily identified in the participants blood smears. SCD patients had a significantly higher proportion of red cells containing HJB (1.5%; IQR 0.7%-3.1%) compared to controls (0.3%; IQR 0.1%-0.5%) (P <0.0001). The AI red cell counts were also higher among the SCD patients (47.4%; IQR 34.5%-66.0%) than the control group (7.1%; IQR 5.1%-8.7%) (P < 0.0001). The intra-observer reliability for assessment of HJB- (r = 0.92; r2 = 0.86) and AI- containing red cells (r = 0.90; r2 = 0.82) was high. The estimated intra-observer agreement was better with the HJB count method (95% limits of agreement, -4.5% to 4.3%; P = 0.579).We have demonstrated the utility of light microscopy in the assessment of red cells containing-HJB and AI inclusions as indices of splenic dysfunction in Nigerian SCD patients. These methods can be easily applied in the routine evaluation and care of patients with SCD to identify those at high risk of infection and initiate appropriate preventive measures.

Assessment of the risk of stroke in children with sickle cell anemia using transcranial doppler ultrasound with imaging in Northwestern Nigeria

Background: Stroke is a major cause of morbidity and mortality affecting about 10% of patients with sickle cell anemia (SCA). Transcranial Doppler (TCD) ultrasonography helps to identify children with SCA who are at an increased risk for stroke. This study aimed to determine the risk of stroke in children with SCA in steady state using Transcranial Doppler with Imaging (TCDI) scan abnormalities and the prevalence of TCDI scan abnormalities among children with SCA in northern Nigeria.&#x0D; Materials and Methods: We conducted acomparative study among 240 persons with SCA who attended a pediatric sickle cell clinic in northern Nigeria and were in steady state condition. We performed a transcranial ultrasound examination and collected blood samples to perform full blood counts and hemoglobin electrophoresis.&#x0D; Results: Abnormal velocities were obtained in 11 (9.3%) children with SCA; while 16 (13.6%) had conditional velocities and 89 (76.7%) of those had normal velocities in one or more of the four vessels studied. Most of the children in the control group 116 (96.7%) had normal velocities and the difference between the groups was not statistically significant (χ 2 = 0.59, p &gt; 0.05).&#x0D; Conclusion: The use of TCDI sonography to predict the risk of stroke should be included in the standard of care in children with SCA in Nigeria and should be included in routine evaluation of disease severity in children with SCA

5588568 ASSOCIATION OF MANNOSE-BINDING LECTIN SERUM LEVEL AND PROMOTER GENE VARIANTS RS7096206 AND RS10031251 WITH FREQUENCY OF VASO-OCCLUSIVE EVENTS IN SICKLE CELL DISEASE PATIENTS; AN EGYPTIAN CROSS-SECTIONAL STUDY

Background: Sickle cell disease (SCD) is a genetic blood disorder involving hemoglobin alterations, in addition to endothelial adhesion, thrombosis and inflammation. Low serum Mannose-binding lectin (MBL) levels can enhance SCD-related blood vessel inflammation as well as reduction of phagocytic activity, leading to sickled erythrocyte accumulation on vessel walls and impaired ability to combat infections. This may contribute to the vaso-occlusive (VOC) episodes. Aim: This work aimed to investigate serum MBL level and MBL promoter gene variants distribution among a cohort of Egyptian SCD patients and to describe their association with VOC events frequencies in the studied patients. Methods: This cross-sectional study included 88 steady-state Egyptian SCD patients aged 6-18 years diagnosed and followed up at Pediatric Hematology Unit, Cairo University Children Hospital. Informed consent was obtained willingly from all subjects or their guardians before enrolment in the study. MBL promoter (-221) (rs7096206) X/Y and (-550) (rs10031251) H/L gene variants were studied by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) technique. Serum MBL level was assayed by ELISA technique with values <500ng/ml considered deficient. Results: The median serum MBL level in SCD patients was 130.9 ng/ml (IQR 71.2-323.9 ng/ml). Patients with serum MBL levels <500ng/ml were significantly older, had longer disease duration and higher rate of VOCs per year, VOCs per lifetime and a higher VOC index (p= 0.039, 0.034, 0.047, 0.040 and 0.008 respectively). Levels <500ng/ml predominated in SS (89.4%) than Sβ groups (70%) (p= 0.023) and in patients with high frequency of VOCs (91.4%) (p=0.008). Serum MBL level correlated negatively with patients’ age, disease duration, hydroxyurea total treatment duration, blood transfusions per lifetime and steady-state HbS level (p= <0.001, <0.001, 0.011, 0.014, 0.031 respectively) but correlated positively with steady-state HbF level (p=0.002). MBL (-221) X/Y both alleles were more associated with (H) allele of MBL (-550) than with L allele (p=0.048), however, with no statistically significant genotype distribution between the studied variants. There was no statistically significant difference in genotype distribution or allele frequencies between MBL (-221) and MBL (-550) in SCD patients regarding serum MBL level, sickle cell type (SS and Sβ), VOCs frequencies or disease severity classes. Conclusion: Serum MBL level rather than studied MBL gene variants is more likely to contribute to the clinical outcomes of SCD; patients with lower MBL levels had a higher rate of VOC per year, per lifetime and a higher VOC index. Meanwhile, the variant genotypes and alleles of the studied MBL promotor gene variants were not associated with MBL deficiency, VOCs, infection events, and disease severity.Further studies to investigate other polymorphisms in MBL gene; MBL exon-1 (codons 52,54 and 57) are recommended. Keywords: MBL, Polymorphism, Sickle cell disease, Vaso-occlusive crises, Egypt

5596911 RETINAL AND MACULAR AFFECTION IN EGYPTIAN SICKLE CELL DISEASE PATIENTS USING SPECTRAL DOMAIN OCT AND OCT-ANGIOGRAPHY: A SINGLE CENTER STUDY

Background: Sickle vasculopathy results from a complex interaction between sickles erythrocytes, white blood cells, platelets, activated endothelium, vasoactive factors and inflammation. Improved life expectancy of sickle cell disease (SCD) patients in recent years has led to emergence of more disease-related complications as ocular complications. Sickle maculopathy, an emerging subtle complication, characterized by local macular thinning secondary to ischemia is not always evident by indirect ophthalmoscope. In an earlier study of an Egyptian SCD cohort using flourscein angiography, we detected proliferative sickle retinopathy (PSR) in 32.5% of enrolled patients; youngest being 15 years old. Discrepancies in reported prevalences among pediatric SCD patients are attributed to different definitions of retinopathy, examining methods used, examiner’s ability, patients’ age and treatment modalities. Spectral domain- Optical Coherence Tomography (OCT) and Optical Coherence Tomography Angiography (OCT-A) are more recent non- invasive tools that allow for early detection of signs of PSR. Aim: This study aimed to assess for retinopathy and maculopathy among an Egyptian cohort of SCD patients using SD-OCT and OCT-A. Methods: This prospective observational cross-sectional study, conducted in Pediatric Hematology Unit, Cairo University Children Hospital, included 80 Egyptian steady state SCD patients (aged 10 years or more) and 20 unrelated healthy age and sex matched subjects as control group. Informed consents were given willingly by all subjects/ guardians before enrollment in the study. SD-OCT (Optovue OCT machine) and OCT-A (Optovue Octa machine) were performed for all enrolled subjects at Opthalmolgy Department, Cairo University. Results: All subjects had normal slit lamp examination. No statistically significant difference was found in best corrected visual acuity (BCVA) of RT eye, Lt eye, slit lamp bio-microscopy or indirect ophthalmoscopy between SCD and control groups (p= 0.059, 0.057, 0.159 & 0.159 respectively). Using SD-OCT, SCD patients had significantly lower mean Rt macular thickness than control group (218.05±19.13 vs 232.1±14.55 micrometer with p-value =0.003). Twelve SCD patients (15%) showed evidence of retinopathy by OCT-A. There was a significant relationship between OCT-A findings and both Rt and Lt macular thickness by SD-OCT (p-value <0.0001 and <0.0001 respectively). OCT-A findings well correlated with Rt BCVA, Lt BCVA, slit lamp bio-microscopy and indirect ophthalmoscope (p-value <0.0001 for all). SCD patients having abnormal OCT-A findings or with macular abnormalities by SD-OCT showed statistically significant higher mean transfusions/ lifetime and longer duration of Deferiprone chelation therapy (p=0.011, 0.014, 0.028 and 0.038 respectively). No significant difference was noted among SCD patients with abnormal vs normal OCT-A findings regarding their studied demographic, clinical or hematologic characteristics. Conclusion: Sickle retinopathy and maculopathy are subtle disease related complications that might affect SCD children and adolescents. SD-OCT and OCT-A are non-invasive useful tools for early timely detection of these complications thus minimizing the risk of sight- threatening retinopathy. Keywords: Retinopathy- Sickle- Egyptian- SD-OCT- OCT-A

Effects of Isoquercetin on Plasma Protein Disulfide Isomerase Activity and Extracellular Vesicle Tissue Factor Antigen/Activity in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) results from a single mutation in the β-globin gene that causes hemoglobin polymerization, red cell sickling, vascular injury, and recurrent vaso-occlusive crises (VOC). Vascular protein disulfide isomerase (PDI) is shown to be critical for thrombus formation by facilitating neutrophil recruitment, platelet activation and vascular fibrin deposition, all processes implicated in sickle vasculopathy. Since plasma PDI is elevated in SCD and cell-surface associated PDI could promote tissue factor (TF) dependent thrombin generation, we investigated the effects of Isoquercetin (IQ) in patients with steady state SCD. Methods: RBC surface associated PDI was quantified by western blotting. Inhibitory effect of IQ on LPS-induced TF expression on sickle monocytes in vitro was determined using flow cytometry. We also measured in vivo the effect of exposure of IQ or placebo for at least 28 days on plasma PDI protein, PDI reductase activity, total plasma extracellular vesicles (EVs), total plasma TF+ EVs, EV-TF-dependent procoagulant activity and markers of coagulation (D-dimer and Thrombin-antithrombin Complexes) using previously published methods. We used plasma from steady state SCD patients participating in a randomized controlled trial (NCT04514510) obtained at baseline and 28 days after exposure to either Isoquercetin 1000mg once daily (n=22) or placebo (n=22). The patients were in steady state, at least 60 days remote from VOC or blood transfusion and on a stable dose of hydroxyurea. The Wilcoxon rank-sum test was used to perform significance testing for differences in the percent change in outcomes between the Isoquercetin and placebo groups. Results: Sickle RBCs had higher cell-surface associated PDI compared to ethnic matched healthy controls (fold change 5.09; p=0.028). In confirmatory experiments with RBC ghosts, extracellular PDI was cell-surface associated. Pretreatment with Quercetin (the active metabolite of Isoquercetin) reduced LPS-induced cell-surface TF expression on sickle monocytes in vitro in a dose dependent manner (Fig 1). Following Isoquercetin administration, there was no change in the percent reduction in PDI inhibitory activity in the IQ group (mean: 6.6, 95%CI [-21.6, 34.9]) compared to placebo group (mean; -31, 95%CI [-94, 32.6], p=0.63). The percent change from baseline in PDI antigen also did not differ between the IQ or placebo groups (IQ mean: 1.2, 95%CI [-11.3, 13.6]; placebo mean: 12, 95%CI [-6, 30.4], p=0.52) (Fig 1). There was no correlation between the percent change in PDI activity and antigen from baseline after treatment with IQ (Spearman's Rho, IQ: r2 =0.28; p=0.20 placebo: r2=0.10; p=0.67). Unexpectedly, total plasma EVs showed no difference between the IQ (mean: 38.4; 95%CI [-4.6, 81.5]) and the placebo group (mean: 9, 95%CI [-18, 37.3]; p=0.27) (Table 1) suggesting that IQ has no obvious anti-inflammatory effects. Moreover, plasma EVs were predominantly derived from RBCs and platelets as published previously by us and others, indicating no membrane stabilizing effect on sickle RBCs and platelets. Inhibiting PDI reductase activity could allosterically reduce TF procoagulant activity, but this hypothesis was untestable because the plasma EV-TF dependent procoagulant activity in this patient population was undetectable, possibly reflecting effective disease control by hydroxyurea. Conclusions: The anti-inflammatory effects of flavonoid quercetin in vitro do not appear to translate into measurable clinical benefits in vivo following short term treatment with IQ in steady state SCD. Trials of higher dose IQ and/or prolonged exposure duration could yield greater inhibition of plasma PDI activity that might reveal detectable clinical antithrombotic effects. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Thrombophilia in Sickle Cell Disease with Isoquercetin: Results of a Fixed Dose, Single Center, Phase II Randomized Clinical Trial

Introduction: Sickle cell disease (SCD) is characterized by elevated markers of hemostatic activation and thrombophilia manifesting as arterial stroke and venous thromboembolism (VTE). Anticoagulation for VTE in SCD is associated with an increased risk for bleeding. In cancer patients at high risk for VTE, the flavonoid Isoquercetin (IQ) lowered D-dimer and soluble P-selectin possibly by inhibiting plasma protein disulfide isomerase. In a murine model of SCD, IQ with Hydroxyurea (HU) reduced platelet accumulation and fibrin deposition following laser-induced injury. Given these results, we tested the hypothesis that IQ would be relatively safe and reduce thrombo-inflammatory biomarkers in steady state SCD patients. Methods: We conducted a phase II randomized, double blind, placebo-controlled study at the NIH Clinical Center. Eligible patients had SCD, were 60 days remote from an acute VOC or blood transfusion and were on a stable dose of HU. Study subjects (n=46) were randomly allocated to receive either Isoquercetin 1000 mg daily (n= 23) or placebo (n= 23) for 28 days and were monitored at the beginning and at completion of study. The primary endpoint of the study was the ELISA measured change in plasma sP-selectin levels after 28 days of placebo or study drug exposure. Analysis was performed on an intention to treat principle. D-dimer levels and Thrombin anti-thrombin (TAT) complex were measured by Immunoturbidimetric assays and ELISA. Statistical analyses was performed using analysis of covariance (ANCOVA) and Wilcoxon rank-sum test. Results: The mean (± SD) age of the study population was 40 years (± 11.57), and 56% were female. The majority had HbSS disease and were receiving HU therapy. A subset (28%) reported a prior history of venous thrombosis (VTE) and four of these were on extended duration oral anticoagulation (Table 1). Baseline characteristics between the two study groups were relatively similar. For the primary endpoint, at the end of study the effect of IQ on the change in plasma sP-selectin levels after adjusting for the baseline was not significant (Figure 1) (IQ mean change= 0.10 ± 6.53 vs. placebo mean change= 0.74 ± 4.54; p= 0.66). Since treatment with HU could confound a treatment effect of IQ in a small sample, we compared the change of sP-selectin among those receiving and not receiving HU but did not find any significant difference (p= 0.67). In a sensitivity analysis we did not find an interaction between HU and IQ treatment (p= 0.48). A similar examination for the potential role of VTE history (n= 5 in IQ and n= 8 in placebo arm) as a confounder did not show a VTE effect on change in sP-selectin (p= 0.68) nor an interaction between VTE history and treatment (p= 0.65). In addition to the primary outcome, we explored the effect of IQ treatment on traditional biomarkers of thrombosis. In contrast to studies in patients with cancer, IQ exposure for 28 days did not have a noticeable effect on change from baseline in either D-dimer levels (IQ mean change= 0.15 mcg/ml ± 0.92 vs. placebo mean change= -0.10 mcg/ml ± 2.11; p= 0.82) or thrombin-antithrombin (TAT) complexes (IQ mean change= 0.09 ng/ml ± 1.60 vs. placebo mean change= -0.05 ng/ml ±1.29; p= 0.57). Conclusion: Short term Isoquercetin exposure in steady state SCD did not appreciably lower plasma sP-selectin but was well tolerated, safe and did not have bleeding side effects. Clinical studies of higher dose IQ with greater exposure duration may yield more measurable effects on antithrombotic activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Specialized Pro-Resolving Mediator (SPM) Profile in Blood from Children with Sickle Cell Disease (SCD): Comparison with Age-, and Race- Matched Controls

Introduction: Specialized Pro-resolving Mediators (SPMs) are lipid mediators, biosynthesized from the n-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and eicosapentaenoic acid (EPA); and the n-6 PUFA arachidonic acid (AA). SPMs are grouped into four families of lipid mediators including resolvins, protectins, maresins and lipoxins; and these mediators play a role in the resolution of inflammation. Inflammation plays a crucial role in the pathogenesis of clinical complications in sickle cell disease (SCD). In addition, multiple studies in SCD have documented decreased levels of anti-inflammatory fatty acids DHA and EPA in blood cells and plasma. We assessed whether the decreased DHA and EPA content play a role in modulation of peripheral blood SPM levels in SCD. Methods: Using targeted liquid chromatography (LC)-tandem mass spectrometry (MS/MS), we investigated plasma SPM profile in children with SCD in steady state (n=45, subjects with confirmed SCD genotypes SS and Sβ0 thalassemia, ages 2-20 years), and compared to that from an age- and race-matched control group (n=24). Analysis also included the SPM pathway markers, prostaglandins, thromboxane and leukotriene and their metabolites. (n-3) PUFA content in total blood cells was measured in both groups using capillary gas chromatography. Results: Consistent with the published results, a significant decrease in (n-3) PUFA content (measured as percent of total fatty acids) was noted in children with SCD (2.14% ± 0.47% in SCD vs 2.39% ± 0.45% in control, mean ± SD, P=0.04). In plasma from both SCD and controls, we identified and quantitated 6 SPMs which included DHA-derived resolvin (Rv) D1, RvD3 and 17R-RvD3; EPA-derived RvE2; and DPA(n-3)-derived RvD5(n-3) and protectin D (PD)-1 (n-3). Median RvD1 level (pg/ml) in SCD plasma was significantly elevated (65 in SCD vs 36 in controls, P=0.002). Levels of other SPMs were not significantly different between the two groups. Multiple SPM pathway markers including mono-hydroxy-PUFAs (EPA-derived 5-HEPE, 12-HEPE, 15-HEPE and 18-HEPE; DHA-derived 4-HDHA, 7-HDHA and 14-HDHA; and AA-derived 5-HETE, 12-HETE and 15-HETE) were present in both control and SCD plasma. Levels (median, pg/ml) of 5-lipoxygenase-derived EPA-metabolite 5-HEPE (212 vs 122, P=0.027) and DHA-derived metabolite 7-HDHA (62 vs 39, P=0.036), and 12-lipoxygenase-derived AA-metabolite 12-HETE (16783 vs 11796, P=0.025) were increased significantly in SCD compared to those from the controls. 15-Lipoxygenase derived pathway markers (15-HEPE and 15-HETE) were not different between the two groups. The cyclo-oxygenase-derived eicosanoids identified and quantitated in both groups included PGE2, PGD2 and TxB2 with a significant increase observed in TxB2 level in SCD plasma (1213 vs 970 pg/ml, P=0.038). Leukotriene (LT) B4 (an AA-derived pro-inflammatory mediator) and lipoxins (AA-derived SPMs), and their metabolites 20-OH-LTB4 and 20-COOH-LTB4 were absent in plasma from both SCD and control. Conclusions: Our results taken together demonstrate that the decreased bioavailability of (n-3) PUFA in children with SCD does not appear to limit SPM production in steady state as documented with normal/increased plasma levels of SPMs and their pathway markers. Whether the SPM profile in circulation is different in SCD pathologies associated with inflammation including vaso-occlusive pain crisis and acute chest syndrome, compared to steady state disease, require further investigation.

Investigating Gene Expression Signatures and Cell-Type Composition Changes in Sickle Cell Disease Using Whole Blood Transcriptomics

Background: Sickle cell disease (SCD) is a devastating Mendelian disease affecting millions of people worldwide. Although SCD is caused by a single-point mutation (Glu6Val) in the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous and systemic, impacting the functions of multiple organs and leading to early mortality. Predicting disease severity and identifying the molecular mechanisms underlying disease pathology and progression remain a challenge. Objective: The goal of this work was to characterize the clinical heterogeneity associated with SCD using transcriptomics. Methods: We integrated publicly available bulk and single-cell RNA sequencing (scRNA-seq) data sets that included samples from pediatric to young adult patients and contained clinical phenotypes to identify molecular signatures of SCD severity and heterogeneity (Table). Our integrative transcriptomics analyses included the following: Identify cell-type clusters and derive novel progenitor signatures from an integrative analysis of the 2 independent scRNA-seq data sets capturing CD34+-enriched or unselected bone marrow (BM) cells from SCD and control samples.Calculate the relative proportions of cell types in whole blood by deconvolution using both the scRNA-seq-derived novel cell-type signatures (dtangle) and publicly available immune cell-type signatures (xCell, EPIC, quanTIseq).Identify signatures (genes, pathways, cell types) associated with disease severity and hemoglobin genotypes using various bioinformatics approaches, including differential gene expression and single-sample Gene Set Enrichment Analysis, among others, and examine relationships of enriched cell types and pathways (queried from MSigDB) in discovery followed by confirmation in validation data sets. Results: Cell-type clusters and gene signatures were generated by combining and integrating scRNA-seq data sets from BM SCD and control samples (Table). Using our fine-mapped cell-type clusters, we found an erythroid progenitor subclass to be depleted, whereas a lymphoid progenitor subclass expanded in SCD BM samples, increasing granularity of the cell types reported by Hua et al. Cell-type deconvolution of bulk transcriptome data using the combined scRNA-seq-derived signatures revealed enriched monocyte relative proportions in acute crisis compared with steady-state SCD samples (β=0.01, P=1.13×10−7) and enriched erythroblast relative proportions in SCD compared with control samples (β=0.02, P=1.10×10−12). Deconvolution using publicly available immune cell signatures showed significant enrichment of neutrophil and macrophage signatures in acute SCD samples (Padj<0.1). Analysis of bulk RNA-seq data indicated a set of highly correlated genes differentially expressed in steady-state SCD and associated with varied pathways, including red blood cell (RBC) morphology, hematopoiesis, anemia, and cholelithiasis. Higher pathway scores were seen in severe (HbSS) genotype samples than in milder (HbSC) genotype samples, consistent with known disease pathology and severity (Figure). Building on results from integrative analyses, we found that interferon and inflammatory response genes were enriched in acute SCD in both discovery and validation data sets. We found significant correlations between monocyte relative proportions and interferon signaling pathway scores, and between erythroblast relative proportions and the pathway scores affecting RBC production, morphology, and pathology and potential SCD comorbidities such as cholelithiasis (Figure). Conclusions: Our integrative scRNA-seq analysis led to robust cell cluster identification to improve deconvolution analysis of bulk transcriptomics data. We found elevated interferon and inflammatory pathway activity in acute SCD samples, consistent with findings in the literature (PMIDs: 34166491, 32764656). Furthermore, we found enriched neutrophils and monocyte signatures in acute SCD, suggesting their role in mediating the innate immune response. These findings provide motivation to characterize the role of neutrophils and monocytes in acute SCD crises. Our integrative transcriptomics analyses showed defective RBC production and maturation in SCD. Using the signatures from this work, we aim to elucidate the heterogeneity within SCD pathophysiology. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of TSP-1, TGF-B1 and Galectin-3 in the Pathogenesis of ACS in Sickle Cell Disease

Sickle cell disease (SCD) is a hereditary blood disorder characterized by painful vaso-occlusive episodes (VOE), acute chest syndrome (ACS), stroke and other clinical complications. Seventy-six percent of the primary and secondary complications of SCD are pulmonary and are strongly associated with hemolysis. ACS is the main acute pulmonary complication of SCD and is characterized by critical lung injury from vaso-occlusion in the pulmonary vasculature, release of inflammatory mediators, and alveolar edema decreasing gas exchange. Endothelial dysfunction, characterized by increased production of inflammatory mediators, increased oxidative stress, and aberrant cellular proliferation via platelet-derived thrombospondin-1 (TSP-1) and transforming growth factor-1 (TGF-B1) is increasingly implicated in pulmonary vascular pathology in other diseases. In addition, a mediator of TGF-B1, galectin-3, has been shown to be induce tissue-specific fibrosis via fibroblast activation in animal disease models. However, the mechanisms by which human pulmonary artery endothelial cell (HPAEC) dysfunction is instigated remains unclear. We hypothesized a novel theory that TSP-1, TGF-B1, and galectin-3 play a causal role in the pathogenesis of ACS in SCD. We set out to test our hypothesis by measuring TSP-1, TGF-B1 and galectin-3 plasma levels in a cohort of patients with SCD with (n=10) and without history of ACS (n=20), and healthy, race and age-matched controls (n=10). Patients with ACS had significantly higher plasma levels of active TGF-B1 (11.34 ng/mL) than those of patients without ACS (5.2 ng/mL) and healthy controls (3.4 ng/mL). Similarly, we found that SCD patients with a history of ACS had significantly higher levels of TSP-1 (642.7 ng/mL) and galectin-3 (8.87 ng/mL) as compared to patients without ACS (285.9 ng/mL and 6.02 ng/mL, respectively) and controls (133.36 ng/mL and 5.2 ng/mL, respectively). We also found a significant correlation between TSP-1 and TGF-B1 levels in non-ACS SCD patients and patients with ACS as well as galectin-3 and TGF-B1 levels in the plasma of all SCD patients combined. Given that platelets are a significant source of TSP-1 and TGF-B1, we next tested whether platelets mechanistically potentiate endothelial cell dysfunction in SCD. HPAECs were co-cultured with platelets isolated from healthy vs steady-state SCD individuals. Platelets in co-culture were quiescent or activated with the agonists 0.1 U/mL thrombin, 25 ug/mL collagen, and 2.5 uM free heme for 6 hours, and HPAEC proliferation was measured via crystal violet staining 48 hours after exposure. We found HPAEC proliferation upon control and SCD platelet exposure to free heme, but SCD platelets exposed to heme induced a more robust response in HPAEC proliferation. In addition, we found that collagen only significantly promoted HPAEC proliferation in the presence of SCD platelets and not control platelets. Activated platelets, in the presence of hemolysis, release large amounts of TSP1 and TGF-B1 that may lead to enhanced endothelial cell proliferation and fibrosis in SCD, possibly via increased response to collagen by activated platelets. We will conduct further experiments to determine whether blockers of TSP-1 or its canonical CD47 endothelial receptor may abrogate aberrant cellular proliferation in SCD. Overall, the roles of TSP-1 and galectin-3 in pulmonary fibrotic injury via the TGF-B1 signaling pathway in SCD need to be further elucidated for therapeutic targeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Iron stores in steady-state sickle cell disease children accessing care at a sickle cell disease clinic in Kumasi, Ghana: A cross-sectional study.

Children with sickle cell disease (SCD) have an increased risk of multiple hemotransfusions and this can predispose them to elevated iron stores. The objectives of the study were to determine the extent of elevated iron stores and the associated risk factors in a population of steady-state SCD children in Ghana. This cross-sectional study was conducted at the pediatric sickle cell clinic at the Komfo Anokye Teaching Hospital. Complete blood count and serum ferritin assay were performed for (n = 178) steady-state SCD children. Descriptive and multivariate logistic regression analysis were performed. Elevated iron stores were defined as serum ferritin levels >300 ng/ml. Statistical significance was considered at p < 0.05. The mean (standard deviation) age of the participants was 9.61 (±4.34) years, and 51% of them were males. About 17% of SCD children had elevated iron stores and receiving at least three hemotransfusions during the last 12 months was strongly associated with elevated iron stores (p < 0.001). History of chronic hemotransfusion increased the odds of having elevated iron store (adjusted odds ratio [aOR] = 11.41; 95% confidence interval [CI] = 3.11-30.85; p < 0.001) but SCD patients on hydroxyurea treatment had reduced-odds of having elevated iron stores (aOR = 0.18; 95% CI = 0.06-0.602; p = 0.006). Moreover, red blood cell (Coef. = -0.84; 95% CI = -0.37, -1.32; p = 0.001), hemoglobin (Coef. = -0.83; 95% CI = -0.05, -1.61; p = 0.04), hematocrit (Coef. = -0.85; 95% CI = -0.08, -1.63; p = 0.03), mean cell volume (Coef. = 0.02; 95% CI = 0.01, 0.03; p = 0.001) and mean cell hemoglobin (Coef. = 0.04; 95% CI = 0.01, 0.07; p = 0.002) could significantly predict serum ferritin levels. The magnitude of elevated iron stores was high among children with SCD in steady-state. Red cell indices could provide invaluable information regarding the risk of elevated iron stores. SCD children who have a history of chronic hemotransfusion or had received at least three hemotransfusions in a year should be monitored for elevated iron stores.