Sickle Cell Disease Patients In Steady State 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.

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

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

Correlation of a Shape Factor and Degree of Saturation of Sickle Cell Red Blood Cells to Vaso-Occulsive Episodes in Sickle Cell Disease

Introduction: Patients with sickle cell disease (SCD) can experience intermittent episodes of vaso-occlusive episodes (VOE) leading to acute pain. Additionally, patients can experience changes in pain over their lifetime such that they can have nociceptive, neuropathic, and opiate induced hyperalgesia, all contributing to the development of chronic pain. Developing a test that can delineate VOE from steady state and also differentiate it from chronic pain continues to pose an elusive problem in SCD. It not only impacts endpoints in clinical studies, it can pose a significant challenge for the patient in terms of timely clinical diagnosis and treatment of pain. Cell Tracking Velocimetry, (CTV), tracks cells on a single cell basis in a very high magnetic energy gradient. This instrument allows us to use the cell's magnetic, v x, and settling velocities, v y, to be transformed into estimates of the oxygen saturation and deformity of the red blood cells, (RBCs). Based on statistical correlations of these measurements to patient status, VOE can be independently identified. Results: Patients were consented in a single center Institutional Review Board, (IRB) approved study. Clinical data including genotype of the patients were collected in 5 clinical states, including healthy donors (0). Steady State no chronic pain ≤ 3 crisis/year (1), Steady state with chronic pain 3 ≤ crisis/year (2), Steady state with no chronic pain > 3 crisis/year (3), Steady state with chronic pain > 3 crisis/year (4), and during acute pain crisis (5). Steady state was defined as no VOC requiring health care intervention for > 4 weeks and no VOE crisis on day of clinic. The patients' samples (n=49; distributed between each state) and clinical state was deidentified for initial laboratory evaluation. The CTV instruments returns two types of data: magnetic velocity and settling velocity. It was demonstrated that the range of the magnetic velocity of the top and bottom 1 percent of the measured velocities (i.e the fastest and slowest moving RBCs) correlates to a patient in VOE, when compared to normal, healthy RBCs and SCD patients in steady state (p > 0.0168). Further, the ratio of the magnetic to settling velocity of the top and bottom 1 percent of these ratios correlates to a patient in VOE, when compared to normal RBCs and SCD patients in steady state (p > 0.0003). These correlations are significant for both RBCs under deoxygenated and ambient air conditions. Discussion: We suggest that these correlations have physical significance since further mathematical modeling of these experimental velocities can be reduced to two factors: a shape factor, fe-x/fe-y, (velocities are proportional to the effective diameter of the cells) and the oxygen-hemoglobin saturation of the RBCs. This ratio of this shape factor can be considered as a measure of how “deformed” the RBC is, while the degree of oxygen-hemoglobin saturation is consistent with the traditional oxygen saturation measurement. Conclusion: While further studies on a larger patient population are warranted, these data suggests that the CTV instrument could potentially provide a biological point of care test to help diagnose VOE. Furthermore, this seems to delineate chronic pain and steady state from acute VOE. A longitudinal study in a consistent patient population would help further delineate and refine this for the specific patient but may offer significant insight over time to correlate the clinical phenotype with a laboratory correlation.

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

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

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

Haematological Changes Associated with Hepatitis B Virus Infection in Individuals with and without Sickle Cell Disease

Background: Hepatitis B virus infection, a major public health problem that primarily affects the liver, may cause reduction in the levels of haemoglobin, haematocrit and in the extreme, could cause aplastic anaemia. The haematological characteristics could be detected with a complete blood count which could provide invaluable information for diagnosis and management of the disease.&#x0D; Aim: To determine the effect of HBV infection on the blood count of individuals with sickle cell disease (SCD) and apparently normal healthy (Non-SCD).&#x0D; Setting: Non-SCD participants were recruited from the community while SCD patients in steady state were recruited from SCD routine clinics.&#x0D; Methods: The study was a cross - sectional study carried out on 1017 non-SCD and 1017 SCD individuals. Haematology Autoanalyzer was used to determine the complete blood count. Granulocyte-to-lymphocyte ratio (GLR), platelet to white blood cell count ratio (PWR) and platelet-to-lymphocyte ratio (PLR) were calculated. ELISA for HBsAg and HBV core antigen IgM antibodies were used to identify participants with HBV.&#x0D; Results: The non- SCD individuals infected with HBV had significantly higher WBC (7.51 ± 5.8 X109/L)) compared to a WBC (6.1 ± 3.4 X109/L) in uninfected individuals (p =0.001). PWR for HBV negative (49.9±28.6) was higher than that for HBV positive participants (41.4±17.6) (p=0.034). Mean platelet volume (MPV) of 9.93 ± 1.1fl in SCD individuals with HBV was significantly higher than 8.30 ± 0.95fl in SCD individuals without HBV (p=.001).&#x0D; Conclusions: PWR and MPV may be useful as surrogate marker for detection of HBV disease progression in apparently normal healthy non - SCD and SCD populations to institute prompt appropriate ancillary investigation and treatment.&#x0D; Keywords: Hepatitis B virus infection; Non-sickle celldisease; blood count; haematologicalparameters; sickle cell disease.

Increased Circulating Copeptin Levels Are Associated with Vaso-Occlusive Crisis and Right Ventricular Dysfunction in Sickle Cell Anemia

Objective: Vaso-occlusive crisis (VOC) is a common clinical manifestation of sickle cell anemia (SCA) and is associated with increased proinflammatory mediators. Copeptin is the C-terminal part of the prohormone for provasopressin and seems clinically relevant in various clinical conditions. Right ventricular (RV) dysfunction significantly appears in SCA patients due to pulmonary hypertension. This study aimed to investigate the association of copeptin levels in VOC patients and evaluate RV dysfunction. Materials and Methods: A total of 108 patients were enrolled in the study. Twenty-eight SCA patients in steady state (30.2 ± 0.9 years), 25 SCA patients in VOC (36.8 ± 11.8 years), and 55 healthy individuals (31.9 ± 9.4 years) with HbAA genotype were included. Clinical, echocardiographic, and laboratory data were recorded. ELISA was used for the determination of serum levels of copeptin. Results: VOC patients had significantly higher copeptin level compared both with controls and SCA subjects in steady state (22.6 ± 13.0 vs. 11.3 ± 5.7 pmol/L, 22.6 ± 13.0 vs. 12.4 ± 5.8 pmol/L, p = 0.009 for both). Additionally, the copeptin level was significantly higher in SCA patients with RV dysfunction than those without RV dysfunction (23.2 ± 12.2 vs. 15.3 ± 9.5 pmol/L, p = 0.024). Multiple logistic regression analysis revealed that high-sensitivity C-reactive protein and copeptin levels were found to be associated with VOC. Conclusion: This study showed that copeptin and hs-CRP levels were increased in patients with VOC, and it was found that RV dysfunction was more common in SCA patients with VOC than in the control group. Copeptin can be considered for use as a potential biomarker in predicting VOC crisis in SCA patients and in the early detection of patients with SCA who have the potential to develop RV dysfunction.

Expression of Netosis Regulators and Correlation with Inflammatory and Coagulation Biomarkers in Sickle Cell Anemia Patients

Introduction: Sickle cell anemia (SCA) is a multi-systemic disease characterized by inflammation and chronic hemolysis. SCA is also associated with an increased risk of venous thromboembolism (VTE), with recent evidence suggesting that coagulation activation could also be involved in the pathogenesis of progressive organ failure in this condition. The generation of neutrophil extracellular traps, also termed NETosis, has been associated with the pathogenesis of VTE, and is also part of the immunothrombosis process. Furthermore, biomarkers of NETosis have been demonstrated in both animal models and SCA patients. NETosis is a complex process that involves the orchestrated participation of several proteins such as peptidyl arginine deaminase (PADI4), neutrophil elastase (ELANE) and myeloperoxidase (MPO). PADI4 mediates histone critulination, an essential step for NETosis, so that inhibition of PADI4 could be considered a potential therapeutic strategy for conditions in which NETosis play a relevant pathogenic role. Although attractive, investment in this strategy requires a more detailed knowledge of the role of PADI4 in pathogenesis of SCA. Objetives: in this study, we aimed to evaluate the expression of PADI4, ELANE, and MPO in steady state SCA patients, and to correlate these results with markers of inflammation and coagulation activation. Methods: mRNA was obtained from isolated neutrophils, and gene expression of PADI4, ELANE, and MPO were measured by qPCR in 54 steady state SCA patients and 40 healthy volunteers matched by age and geographic origin. Gene expressions levels were then correlated with inflammatory mediators and D-dimer. Results: no significant difference could be observed in the expression of PADI4 (0.55 vs 0.49; P = 0.42) and MPO (0.29 vs 0.39; P = 0.88) between healthy volunteers and patients respectively. In contrast, ELANE was up-regulated when patients compared with controls (1.46 vs 0.16; P <0.0001). PADI4 expression did not correlate with any biomarker of inflammation or coagulation (P>0.05). Interestingly, both ELANE and MPO were positively correlated with D-dimer (R=0.4; P=0.004 and R=0.32; P=0.004, respectively), and Von Willebrand factor (R=0.27; P=0.04 and R=0.30; P=0.04, respectively). ELANE was also correlated with TNF-α (R=0.30; P=0.03). Conclusion: gene expression of ELANE, but not PADI4 is up-regulated in steady state SCA patients, with both ELANE and MPO presenting significant associations with D-dimer and other biomarkers of inflammatory activation. Additional studies are ongoing to evaluate PADI4 expression and activity, both in steady state and during acute episodes in SCA. A more detailed evaluation of the role of PADI4 in the pathogenesis of SCA is warranted for the proper design of clinical trials based on PADI4 inhibition. DisclosuresNo relevant conflicts of interest to declare.

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

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

Pulmonary Hypertension in Sickle Cell Disease: The Role of Activated Blood Coagulation and Inflammation.

Introduction: Pulmonary hypertension (PHT) is a common complication of sickle cell disease (SCD). The pathogenesis of PHT in this setting is likely multifactorial. There is an abundance of evidence to suggest that the coagulation system is activated in patients with SCD. In addition, autopsy series have shown that an in situ thrombotic arteriopathy is seen in the majority of SCD patients with PHT. The purpose of this study is to determine whether markers of thrombin generation, platelet activation and inflammation are associated with PHT in SCD patients.Methods: This cross-sectional study was performed using a cohort of patients followed at the Sickle Cell Clinic at UNC-Chapel Hill. The pulmonary artery systolic pressure (PASP) was determined with a Doppler echocardiogram by applying the modified Bernoulli equation (PASP = 4V2 + right atrial pressure). Pulmonary hypertension was defined using an age-, sex- and BMI-adjusted reference range. Laboratory studies, including a complete blood count, soluble CD40 ligand [sCD40L] (a marker of inflammation and platelet activation) and markers of thrombin generation (thrombin-anti-thrombin complex [TAT], prothrombin fragment 1+2 [F1+2], and D-dimers) were obtained on all SCD subjects and on six healthy, race-matched control subjects without SCD. Wilcoxon Rank Sum Tests were used to compare median values in SCD patients with and without PHT, as well as between SCD patients and the control individuals.Results: Of the 76 subjects with SCD (SS = 56, SC = 9, Sβ0 = 4, Sβ+ = 7), PHT was found in 26 (34%). Compared with SCD patients without PHT, those with PHT had higher WBC counts (10.4 X 103/μL vs. 8.7 X 103/μL; p = 0.05). In addition, while PHT patients had consistently higher levels of such parameters as: D-dimers (1603 ng/mL [FEU] vs. 1233 ng/mL [FEU]; p = 0.3), TAT (7.05 μg/L vs. 5.7 μg/L; p = 0.8) and F1+2 (1.15 nmol/L vs. 0.9 nmol/L; p = 0.3), they failed to achieve statistical significance. On the other hand, the sCD40L level was lower (6.7 ng/mL vs. 7.6 ng/mL; p = 0.6) in the PHT group, although this difference was also not significant. In a second analysis, SCD patients had significantly higher levels of D-dimers (1251.5 ng/mL [FEU] vs. 318 ng/mL [FEU]; p = 0.02), sCD40L (7.3 ng/mL vs. 2.8 ng/mL; p = 0.0003), WBC count (9.2 X 103/μL vs. 7.1 X 103/μL; p = 0.02) and platelet count (399 X 103/μL vs. 251 X 103/μL; p = 0.002) when compared to control individuals. The TAT and F1+2 levels were also higher in SCD patients, although the differences were not significant.Conclusion: Compared to controls, SCD patients have increased thrombin generation, platelet activation and inflammation. In our cohort, SCD patients with PHT have higher levels of various markers of thrombin generation compared to patients without PHT. The lack of statistical significance is likely a result of the baseline elevation of these markers in SCD patients in steady state and as such, much higher increases in the levels of these markers and/or much larger sample sizes would be required to reveal a significant difference between both groups. This evidence of further activation of coagulation in SCD patients with PHT provides a rationale for clinical trials exploring the use of anticoagulation therapy in these patients.

Evaluation of Von Willebrand Factor and other Coagulation Homeostasis Profile of Patients with Sickle Cell Anaemia attending a Tertiary Hospital at Enugu, Nigeria

Background:&#x0D; Sickle cell anaemia (SCA) is a genetic disorder that has been shown to facilitate certain forms of coagulopathy. However, there is a paucity of data that demonstrated these features in Nigerian Sickle cell anaemic patients.&#x0D; The objective of study:&#x0D; This prospective study aims to determine the plasma level of vWF, APTT, PT, platelet count and haematological parameters of SCA patients in steady state in comparison with apparently healthy individuals attending the University of Nigeria Teaching Hospital, Enugu, Nigeria.&#x0D; Materials and Methods:&#x0D; A total of 85 participants were enrolled in this study. This comprised of 35 homozygous SCA in steady-state as test subjects (HbSS) and 50 apparently healthy individuals of Hb AA as the control group. Blood samples were collected from all participants; both PT and APTT were analysed using Quick and kaolin methods, respectively., vWF was assayed using Enzyme-Linked Immunosorbent Assay (ELISA). Haematological parameters were analysed through the use of an automated haematology analyser.&#x0D; Results:&#x0D; There was significant elevation of mean±SD of vWF, APTT and platelet count among SCA patients (52.53 ± 1.45 iu/dl), (48.40 ± 4.99 s) and (287.68 ± 21.43 x109/l) when compared with the mean value in control group (40.16 ±1.50 iu/dl), (33.88 ± 1.34 s) and (219.67 ± 13.9 x109/l) (p&lt; 0.05). However, there were significant decrease in PCV, haemoglobin concentration and red blood cell count among SCA patients, (24.00 ± 0.82 l/l), (7.89 ± 0.24 g/l) and (2.97 ± 0.8 x 1010/l) when compared with control group (40.95 ± 0.73 l/l), (12.44 ± 0.26 g/l) and (4.81 ± 0.11 x 10/l) (p˂ 0.05) respectively. In addition, significant elevation of mean ±SD MCH and MCHC with mean values of (26.63±0.93 pg) and (33.28 ± 0.52 g/l) among subjects when compared with control groups (25.68± 0.47 pg), (30.30 ± 0.18 g/l) (p˂0.005). However, there were no significant differences in WBC, PT and MCV in both study groups.&#x0D; Conclusion:&#x0D; Findings from this study demonstrated prolonged coagulation profile, thrombocytosis and leucocytosis among SCA patients in steady state.

Vitamin D Deficiency and Its Association With Anemia and Blood Transfusion Requirements in Nigerian Adults With Sickle Cell Anemia

Background Vitamin D supplementation has been shown to improve hemoglobin levels in patients with sickle cell anemia (SCA). However, very little is known about the prevalence of its deficiency, its role in hemolysis, and its effects on transfusion requirements in SCA, hence this study. Patients and Methods Serum level of vitamin D was determined in 50 SCA patients in steady state, 50 SCA patients in hemolytic crises, and 50 in normal HbAA individuals. All were 18 years or older and were age- and sex-matched. In addition, PCV and hemoglobin (Hb) concentration, absolute reticulocyte count, uric acid, total and conjugated bilirubin, creatinine, lactate dehydrogenase (LDH), and estimated glomerular filtration rate (eGFR) were also assessed in all subjects and controls. Baseline sociodemographic, clinical, and anthropometric data were also recorded. Results The prevalence of vitamin D insufficiency and deficiency was found to be 18% and 79%, respectively. The mean serum levels of vitamin D were 18.84 ± 6.86 ng/mL and 17.58 ± 6.23 ng/mL in the steady-state and the hemolytic crises SCA groups, respectively. Transfusion requirements were found to be higher in SCA patients with lower vitamin D levels. Unlike those in crisis who received one or more units of blood transfusion, 42% of those in the steady state did not receive blood transfusion over the same period of 12 months. Hemoglobin levels were, however, found to predict reduced vitamin D levels. Conclusion Vitamin D deficiency (VDD) is highly prevalent among adults with SCA and those with lower vitamin D levels are more anemic and hence may require more blood transfusions.

Plasma Folate Levels in Acutely Ill and Steady State Pediatric Sickle Cell Disease Patients in Ghana.

BackgroundIndividuals with sickle cell disease (SCD) are susceptible to infective conditions that predispose them to hemolysis and anemia. Folic acid is recommended as a preventative measure against anemia in SCD patients; however, there is scarce literature on the implications of this practice.Patients and MethodsPlasma concentrations of folate were measured in acutely ill pediatric SCD patients presenting with malaria or bacteremia and compared with those of SCD patients in steady state, or acutely ill non-SCD patients with confirmed malaria.ResultsThe proportion of individuals with high (>45.3 nmol/L) folate concentrations was 29.5% (13/44), 18.2% (8/44), 33.3% (6/18), and 0% in the SCD-malaria, SCD steady state, SCD bacteremia, and the non-SCD malaria groups, respectively. The proportion of SCD patients with high folate levels did not vary significantly at steady state and during confirmed malaria (p = 0.216), and during acute bacteremia (p = 0.20). The median (interquartile range) plasma folate levels were 34.50 (24.40–52.00 nmol/L), 33.40 (15.83–60.85 nmol/L), 30.85 (24.68–39.65 nmol/L), and 13.30 (10.03–17.18 nmol/L), respectively, in the SCD malaria, SCD bacteremia, SCD steady state, and the non-SCD malaria sub-groups. The median folate levels of SCD steady state, SCD malaria, and SCD bacteremia sub-groups differed significantly (p < 0.0001) when compared with non-SCD patients, but the levels in the SCD bacteremia and malaria groups were not significantly different from the SCD steady state group.ConclusionElevated levels of plasma folate were found in a high proportion of pediatric SCD patients. The implications of such elevated folate levels in pediatric SCD patients are unknown but may suggest a need for review of current recommendations for prophylactic doses of folic acid in SCD patients.

Sickle cell avascular necrosis: Prevalence and clinical profiles in a tertiary hospital northwestern Nigeria

Acute painful crises are the hallmark of Sickle Cell Anaemia (SCA). However, chronic daily pain also occurs in SCA patients with a high frequency. Avascular Necrosis (AVN) of the femur is an important cause of chronic pain and adversely affects their quality of life. The aim is to determine the prevalence of AVN among SCA patients in Zaria and describe some of its clinical and laboratory features. A cross-sectional study of 58 SCA patients in steady state. Data on gender, age, presence of radiologically diagnosed AVN, number of pain crises, blood transfusions in the previous 12 months and laboratory parameters were collated. Data were analyzed using JASP version 0.11.1.0. A critical level of α of 0.05 was set. Females constituted 40 out of 58 (69.0%) of the study participants. The median age was 23 (19.8, 28.0) years. AVN was present in 6 out of 58 (10.3%). There was no relationship between gender and AVN (FET, p=1.00, OR=0.889 95% CI 0.147, 5.359). There was no age difference between patients with AVN and those without AVN (29.92 vs 29.45, MWU=153.500, p=0.956). Patients with AVN had lower mean rank HCT levels (24.08 vs 30.13, MWU=123.500, p=0.414), higher pain episodes (31.67 vs 29.25, MWU=169.000,p=0.747), number of blood transfusions (33.42 vs 29.05, MWU=179.500, P=0.549) and platelets (34.00 vs 28.98, MWU=183.000, p=0.499) compared to those without AVN. Avascular necrosis is common among SCA patients in Zaria. These patients have more blood transfusions and bone pain episodes compared to those without AVN.

Investigation of Lipid Profile and Clinical Manifestations in SCA Children.

Introduction Clinical complications in sickle cell anemia (SCA) are heterogeneous and involve several molecules. It has been suggested that SCA individuals present a dyslipidemic phenotype and that lipid parameters are associated with severe clinical complications, such as pulmonary hypertension. We sought to investigate associations between lipid parameters and clinical manifestations, as well as other laboratory parameters in a population of pediatric SCA patients. Methods Our cross-sectional evaluation included 126 SCA patients in steady state and who were not undergoing lipid-lowering therapy. Hematological and biochemical parameters were characterized, and previous clinical manifestations were investigated. Results Total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels were increased in patients with a previous history of pneumonia, which also positively correlated with HbS levels. Decreased LDL-C levels were also associated with leg ulcers and anemia. Elevated high-density lipoprotein cholesterol (HDL-C) levels were associated with pain crises, increased viscosity, and decreased hemolysis. Several studies have determined that lipids play a role in the vascular impairment seen in SCA, which was corroborated by our findings. Conclusions In sum, our results suggest that total cholesterol, HDL-C, and LDL-C levels are associated with hemolysis and anemia markers and, most importantly, with clinical complications related to vasculopathy in SCA.

Burden and Risk Factors of Chronic Kidney Disease in Children with Sickle Cell Anaemia Aged 5 – 16 Years at the University Teaching Hospital, Lusaka - Zambia

Background: Improved medical care has led to the improved life expectancy of sickle cell anaemia (SCA) patients hence complications associated with SCA such as chronic kidney disease (CKD) are being seen more frequently. Globally, nephropathy of varying severity occurs in 5 to 18 % of the SCA population across all age groups with a third of the adults proceeding to develop CKD while over 30 % of paediatric SCA patients have CKD in Africa. The mortality rate in SCA patients CKD is high. This study sought to determine the prevalence and risk factors of CKD in SCA, information that was not available in Zambia prior to this study. This information will guide in targeting and timing of screening for CKD in SCA in children in our population. Objectives: To determine the prevalence of haematuria, proteinuria, abnormal estimated glomerular filtration rate (eGFR), CKD, and risk factors of CKD among the steady-state SCA patients aged 5 to 16 years at the University Teaching Hospital (UTH), Lusaka. Methodology: This was a prospective cross-sectional study of 197 children aged 5 to 16 years with SCA at the UTH - Lusaka conducted from August 2014 to July 2015. Demographic and clinical data were collected using a structured questionnaire. Urine and blood samples were used to determine the urine albumin creatinine ratio (ACR) and full blood count /blood biochemistry respectively. CKD was defined and determined using the Kidney Disease Outcome Quality Initiative 2012 guidelines employing urine ACR, dipstick urinalysis and eGFR. In this study, spot urine ACR and dipstick urinalysis were done and repeated three months later if initial tests were abnormal. Data was analysed using SPSS version 21. Chi-square and t-test were used to compare proportions between groups. Relation between study variables and CKD were examined using logistic regression. Results: The mean age of the participants was 9.6 years (SD ±3.6). Male to female ratio was 1:1. The median age at diagnosis of SCA was 22 months (IQR = 44). The prevalence of haematuria, proteinuria and CKD among the study participants was 14.2%, 36% and 36 % respectively. Low haemoglobin and elevated mean corpuscular volume (MCV) were associated with CKD-AOR 0.62, 95% CI; 0.46-0.84 and 1.04, 95% CI; 1.01 – 1.08 respectively. Recurrent admissions (due to VOCs, severe anaemia and febrile illness) were also risk factors associated with CKD- AOR 0.52, 95% CI; 0.27-0.98. CKD was not associated with age at enrolment, sex, age at diagnosis of SCA, recurrent Vaso-occlusive crisis (VOCs) or abnormal liver function tests. Conclusion: The prevalence of CKD among the SCA patients at UTH- Lusaka is high (36%) with lower Haemoglobin, elevated MCV and recurrent admissions being risk factors for developing CKD. SCA patients should be screened for CKD routinely at least once a year. Interventions such as the early introduction of hydroxyurea, proactive blood transfusions and ACE inhibitors can reduce the risk of CKD and its progression to end-stage renal disease.

Ultrasound findings in the gallbladder of sickle-cell patients: A cross-sectional Study in Enugu, Nigeria

Background: Sickle‑cell disease (SCD) causes chronic and recurrent hemolysis which is a recognized risk factor for gallbladder (GB) disease. The prevalence of GB disease in SCD is high and increases with age. Cholelithiasis and cholecystitis are relatively common GB diseases in SCD. They are important causes of acute abdominal pain in SCD and also increase morbidity. Ultrasound is a cheap and noninvasive means of evaluating the GB; it has a high degree of accuracy and is widely available. Aims: The aim of this study is to sonographically evaluate the GB of SCD patients in steady state at the University of Nigeria Teaching Hospital (UNTH), Ituku‑Ozalla, Enugu, and to determine the prevalence of specific GB diseases in SCD patients. Materials and Methods: A prospective cross‑sectional study of 130 known SCD patients attending sickle‑cell clinics at UNTH and 130 controls. Ultrasound of the GB was done after at least 6 h fast, using a Dp2200 mindray mobile ultrasound machine equipped with a 2.5–5 MHz convex probe. The data were analyzed using the SPSS software version 16. Results: The GBs of 130 consecutive SCD participants and 130 control group were sonographically evaluated. The prevalence of cholelithiasis in SCD was 10.8%, whereas the prevalence of cholelithiasis in the control group was 3.1%. No other GB diseases were seen. The mean dimensions of theGB for the study group were length (65.78 ± 9.85 mm), width (29.15 ± 6.01 mm), depth (25.58 ± 5.81 mm), wall thickness (1.59 ± 0.74 mm), and volume (26.52 ± 11.18 cm3). Conclusion: The prevalence of cholelithiasis in sickle cell disease in southeast Nigeria is 10.8% while the prevalence of cholelithiasis in the control population is 3.1%. Keywords: Cholecystitis, cholelithiasis, gallbladder, sickle cell disease, ultrasound

Portal hypertension evolving from sickled hepatopathy: Could hepatic venous Doppler ultrasound be beneficial in its evaluation?

PurposeSickle cell intrahepatic cholestasis involves sickling within hepatic sinusoids leading to vascular stasis and localized hypoxia resulting in ballooning of the hepatocytes causing a direct back pressure effect with resultant intracanalicular cholestasis. Vascular stasis may ultimately lead to portal hypertension. We proposed to document findings suggestive of portal hypertension evolving from hepatopathy in steady-state sickle cell disease (SCD) patients using hepatic venous Doppler ultrasound. MethodsThis is a prospective case series of 6 SCD subjects in steady-state (median age, 30 years; range, 19–43), comprising of 3 males and 3 females, who underwent a routine Doppler ultrasound evaluation of their hepatic veins and were discovered to have an abnormal biphasic waveform pattern. Venous blood was obtained from all subjects to evaluate for P-selectin, homocysteine, foetal haemoglobin, haematocrit levels, white cell and platelet counts. Doppler ultrasound was also carried out on all subjects to evaluate for the hepatic waveform, right renal artery RI and PI along with the hepatic artery velocities. ResultsAll the 6 subjects had reduced haematocrit (median value of 21.5%; range, 18–25%) and some degree of renal dysfunction (plasma cystatin-C ranged from 1.6 to 12.2 mg/L). Elevated white cell count, hyperhomocysteinemia, reduced SpO2(<94.0%) and reduced estimated GFR (eGFR < 90 ml/min) was also noted in 4 subjects (66.7%). Similarly, 4 subjects (66.7%) had elevated RI in the right kidneys while 3 subjects (50.0%) had elevated PI in the right kidney. ConclusionDoppler ultrasound Hepatic vein waveform analysis may be a useful examination in the evaluation of patients with SCD as it may elicit feature of portal hypertension. Further studies are suggested to confirm this in a larger population of SCD patients using the gold standard.