Steady-state Sickle Cell Disease Research Articles

Assessment of Bone Marrow Function in Sickle Cell Anaemia Patients Using Corrected Reticulocyte Counts

Introduction: Chronic hemolysis occurs in sickle cell anemia as a result of recurrent sickling and other abnormalities of the red blood cells including eryptosis. Exuberant reticulocytosis is anticipated to partially compensate for the resultant anemia. Sickle cell anemia patients may also have aplastic crisis, bone marrow (BM) infarction and erythropoietin deficiency which could lead to reticulocytopenia despite the anemia. High degree of reticulocytosis among asymptomatic infants with sickle cell anemia has been associated with an increased risk of death or stroke during childhood. Assessment of BM function in sickle cell anemia is important due to potential complications associated with both under-activity and hyperactivity. This study aimed at evaluating the erythropoietic function of the BM in steady state sickle cell anemia using corrected reticulocyte counts.Methods: This study was carried out at the hematology clinic in the University College Hospital, Ibadan. HbSS patients in steady state were recruited from the hematology clinic. Local ethical committee approval was obtained and all participants gave written informed consent. Patients with M. tuberculosis, Hepatitis B, HIV and P. falciparum infection were excluded. Peripheral blood samples were analyzed using Sysmex Ki-X21 for complete blood count (CBC) and standard point of care for serum electrolytes and liver function tests. The glomerular filtration rates were calculated using the Cockcroft-Gault formula. Reticulocyte counts were determined manually using fresh samples from K2 EDTA bottles and methylene blue stain. Two drops of stain were mixed with two to four volumes of anticoagulated blood and incubated at 37ºC for 15 minutes. Afterwards, the cells were re-suspended and blood films were made. Corrected reticulocyte count and reticulocyte production index were calculated. Participants were categorized according to corrected reticulocyte counts of greater than or less than 2.5%. Univariate and multivariate analyses were performed to determine variables associated with corrected reticulocyte count <2.5%.Results: 92 HbSS patients were recruited with a mean (SD) age of 19.6 (5.8) years. There was no correlation between age and eGFR (p-value: 0.227). Median (range) reticulocyte count, corrected reticulocyte count and reticulocyte production index were 5.5 (0.5 - 29.9), 3.3 (0.1 - 17.1) and 1.7 (0.2 - 8.6) respectively. 40 (43.5%) patients had corrected reticulocyte count <2.5% and 52 (56.5%) had a corrected count >2.5%. Those corrected reticulocyte count <2.5% were older (p: 0.013), taller (p: 0.041) and had higher aspartate transaminase (AST) levels (p: 0.006) than those with corrected counts >2.5% (Table 1). CBC parameters were not different when compared between both groups. Results of multivariate logistic regression analysis carried out showed that only AST was independently linked with corrected reticulocyte count <2.5% (R2: 0.172, p-value: 0.001) (Table 2).Table 1Factors Associated with Low Reticulocyte CountCorrected count<2.5%Corrected count>2.5%p-ValueAge (Mean, SD)21.4 (6.3)18.4 (5.0)0.013Gender (N, %)Male22 (55.0)28 (53.8)0.912Female18 (45.0)24 (46.2)Height (Mean, SD)1.6 (0.1)1.5 (0.1)0.041BMI (Mean, SD)18.7 (3.1)18.7 (3.0)0.753GFR (Mean, SD)64.3 (37.7)66.4 (29.3)0.453Bilirubin (Mean, SD)1.7 (1.1)1.9 (2.6)0.674AST (Mean, SD)22.5 (13.5)14.5 (6.6)0.006ALT (Mean, SD)13.4 (7.7)14.4 (11.1)0.876Table 2Independent Predictors of Corrected Reticulocyte Count <2.5%or95% CIp-ValueAge1.080.97 - 1.210.169Height19.80.11 - 366.100.259AST1.101.04 - 1.170.002Hemoglobin1.000.97 - 1.020.872R2: 0.172, p: 0.001Conclusion: Despite corrected reticulocyte count <2.5% in about half of the patients, there were similar hematological parameters and eGFR in both groups of patients. AST is a marker of hemolysis and low ALT rules out hepatic involvement. Since only 17.2% of the variability in BM response as assessed by corrected reticulocyte count could be accounted for by variables included in this study, there is a need to further evaluate the BM function of sickle cell patients to establish the causes of corrected reticulocyte count <2.5% in the setting of anemia, having ruled out erythropoietin as well as iron, folate or cobalamin deficiencies. This will aid the development of a functional algorithm for the individualized management of sickle cell disease patients with anemia. DisclosuresNo relevant conflicts of interest to declare.

Hypersegmented Neutrophil Percentage Using Automated Digital Cell Morphology: A Simple Laboratory Parameter to Monitor Hydroxyurea Therapy in Sickle Cell Disease Patients

Fetal hemoglobin (HbF) induction with hydroxyurea (HU) remains the only specific pharmacological treatment for sickle cell disease (SCD) patients. Compliance is key to achieve high HbF levels, but assessing patient adherence to long-term treatment is difficult. Since patient interviews are often unreliable, most hematologists rely on mean corpuscular volume (MCV) and HbF levels to monitor compliance, but red blood cell indices change slowly. Hypersegmented neutrophils (HN) have been for long recognized in patients taking HU, and automated digital cell morphology platforms allow routine peripheral blood smears to be conveniently photographed and stored for analysis. We have designed a protocol to determine the percentage of circulating HN and have studied how this parameter compares with others commonly used in clinical practice during HU therapy, such as HbF, MCV, and reticulocyte count.We collected blood samples from 38 patients with SCD in steady state, receiving an average dose of 20.5mg/kg/day of HU (range 10.2-33.3) at our Hematology outpatient clinic. Complete blood counts and HN percentage determinations were performed on a Sysmex XE-5000/Cellavision DM96 equipment. HbF levels were determined by HPLC (Bio-Rad). Statistical analysis was performed using GraphPad Prism 6.0 software. Standard protocol for image acquisition initially recorded 100 cells per smear and HN count was performed independently by two experienced laboratory personnel using the same image database, by counting cells with 5 or more nuclear segments. To increase precision due to the relatively low numbers of neutrophils in some samples, analysis was subsequently improved by acquiring 300 images per smear aiming to picture 100 neutrophils.Mean hemoglobin level in our population was 8.6±1.3g/dL (mean±SD), MCV 103.8±14.2fL, reticulocyte count 222,950±129,090/uL, and HbF was 13.1±7.8% (range 2.1-30.9%). HN percentage as determined with 100 images per smear displayed only borderline correlation with HbF levels (P =0.094), but acquisition of 300 images per smear yielded over 90 neutrophil images in 35/38 samples. Average HN percentage was 14,5% (range 2,0-45,0%) and correlated positively with HbF (r=0.4172, P =0.009) and MCV (r=0.4301, P =0.007). As expected, HbF also correlated with MCV (r=0.5777, P =0.0001) and reticulocyte count (r=-0.489, P =0.003). Despite our limited number of patients, ROC curve analysis showed that HN percentage had an area under curve of 0.7241 to detect patients with HbF>20% (P =0.047). Patients with more than 6 segments per neutrophil were also more likely to have higher HbF. Average daily HU dose did not correlate with HbF or any of the parameters analyzed.Lack of correlation between dose and HbF supports that medication adherence may indeed be suboptimal in the study population, but that may also be caused by individual differences in HU metabolism. While MCV still displayed the strongest correlation with HbF in our study, HN percentage performed similarly, with higher counts associated with higher HbF. Neutrophils have a much shorter half-life than erythrocytes, with an estimated half-life of hours rather than the 20 days calculated for sickle red blood cells. Therefore, digital cell morphology analysis enables clinical laboratories to determine HN counts that can change more quickly than HbF, MCV, or reticulocyte counts during HU therapy. Monitoring the number of HN may allow more timely assessment of compliance in patients starting HU or in those in need for HU in combination with sporadic blood transfusions, since neutrophil hypersegmentation should not be affected by changes in red blood cell mass. Further studies should investigate HN percentage as a potential surrogate marker of response to HU and of patient compliance.Financial support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Heme Concentration As a Biomarker of Sickle Cell Disease Severity: Its Role in Steady-State and in Crisis Patients

Background: Sickle cell disease (SCD) is a heredity group of anemia characterized by hemolysis, chronic inflammation, and vaso-occlusive/painful crisis. The heme is a product of erythrocytes' lyses and is increased in hemolytic diseases.Objectives: To investigate associations between hematological/biochemistry biomarkers and total plasma heme levels between SCD patients groups (in crisis and in steady-state) and healthy controls. To identify molecules related to hemolysis, inflammation, hepatic dysfunction, renal and lipid metabolism.Methods: We evaluated a total of 125 SCD steady-state patients, 22 SCD in crisis patients, and 32 healthy individuals age- and sex-matched with patients groups. Hematological analyses were performed by automatic cell counter, and hemoglobin profile by HPLC. Biochemistry analyses of inflammatory and infection markers, as well as lipid, hepatic, and kidney metabolism markers were investigated by immunochemistry assays. Plasma concentration of total free heme was measured by QuantiChrom Heme Assay Kit.Results: SCD patients groups (steady state and crisis) had higher heme concentration when compared to healthy individuals (p < 0.0001). However, significant difference of heme level was not finding in the comparison between SCD in steady state and SCD crisis patients groups. Biomarkers analyses of steady-state SCD patients showed negative correlation between heme levels and: red blood cell (r = -0.36, p<0.0001); hematocrit (r = -0.38, p <0.0001); hemoglobin (r = -0.34, p <0.0001); and HDL-C (r = -0.42, p <0.0001). Heme level showed positive correlation with: platelets (r = 0.35, p <0.0001); lactic dehidrogenase (r = 0.40, p <0.0001); reticulocytes count (r = 0.19, p =0.04); monocytes count (r = 0.36, p <0.0001); HbS concentration (r = 0.54, p <0.0001); total proteins (r = 0.22, p =0.01); AST (r = 0.41, p <0.0001); ALT (r = 0.18, p= 0.04); serum iron (r =0.21, p =0.03); total cholesterol (r = 0.23, p =0.01); LDL-C (r = 0.22, p= 0.02); VLDL-C (r = 0.65, p <0.0001); and triglycerides (r = 0.63, p <0.0001). In steady state SCD patients there was no difference of clinical manifestations history and heme levels.Conclusions: The finding of similar heme concentration between steady state and crisis SCD patients may be explained by the hyperhemolysis phenomenon, showing that even in steady-state, these patients continue to have hemolysis and generate heme and reactive oxygen species. It was also shown that high concentration of free heme increases hemolytic and inflammatory biomarkers, such as LDH, bilirubin's, reticulocytes count, and lipids, contributing to a severe clinical modulation of SCD. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia Reoxygenation Treatment Induces Platelet Hyperactivity and Relieves Calpain-1-Mediated Inhibition of Platelet Aggregation in a Mouse Model of Severe Sickle Cell Disease

Introduction: With an estimated 15 million patients and no drug that addresses its etiology, sickle cell disease (SCD) remains an area of unmet need. Vaso-occlusive pain crisis (VOPC), the hallmark of SCD, is initiated by sickle RBCs (sRBCs) recruiting leukocytes and platelets to potentiate vessel occlusion. ADP released by sRBCs is a potent activator of platelets, and sickle cell patients are known to have activated platelets in circulation both at steady state and during VOPC. However, the mechanism underlying platelet dysfunction in SCD is not fully understood. Platelet activation mediated by the protease activated receptors (PAR1 and PAR4 in humans, PAR3 and PAR4 in mice), triggers PLC-β activation resulting in calcium mobilization. The increased calcium flux leads to activation of GPIIbIIIa/aIIbb3, GP1b, and P-selectin involved in platelet aggregation, adhesion, and rolling. Prior evidence has established a role of the calcium-activated cysteine protease, calpain-1 in platelet activation. Washed platelets from calpain-1 knockout C57BL/6 mice demonstrated impaired platelet aggregation. However, due to the critical contribution of sRBCs to platelet dysfunction in SCD, whole blood (impedance) aggregometry represents a physiological assessment of platelet aggregation.Methods: Townes SCD mice (SS) were backcrossed with calpain-1 knockout (CKO) mice to generate SCD mice lacking calpain-1 (SSCKO). Humanized mice (AA) were used as controls. Using flow cytometry, we evaluated in vivo platelet activation following stimulation with ADP and Thrombin GPRP. Platelet counts were obtained via ADVIA 120 and flow cytometry. For platelet aggregation, 500 μL of blood was harvested from the vena cava of AA, SS, SSCKO, and CKO mice. Whole blood aggregation in response to PAR4 stimulation was assessed using the Roche Multiplate Analyzer. A separate group of mice were challenged with hypoxia/reoxygenation (H/R) treatment (3 hours of 10.5% O2, followed by 4 hours of 21% O2) prior to platelet aggregation testing. SCD mice are characterized by tissue infarcts suggestive of thrombus formation. To examine whether H/R treatment induces formation of fibrin thrombi, we harvested brain, lungs, heart, kidneys, liver, and spleen following blood collection, and performed histology.Results: Compared to AA, SS and SSCKO mice are thrombocytopenic. Similar to Berkeley, Townes mouse platelets are activated in vivo, demonstrated by activated GPIIbIIIa on circulating platelets. At steady state, PAR4 agonist-induced platelet aggregation is similar in AA and SS mice (64 U v. 53 U, n = 6-10/group, p = 0.3). As depicted in Fig 1., SSCKO mice show significantly reduced platelet aggregation compared to SS mice (13 U v 53 U, p<0.001, n = 6-10 per group). As expected, H/R treatment induces platelet hyperactivity in SS, but not AA mice (71 U v. 59 U, p = 0.04, n = 6/group). Interestingly, the H/R-induced platelet hyperactivity partially relieves the calpain-1-mediated inhibition of platelet aggregation in SSCKO mice at steady state (Fig 2) (53 U v. 13 U, p<0.001, n = 6-8/group). Furthermore, CKO mice do not show defective platelet aggregation at both steady state, and following H/R treatment, indicating an important role for sRBCs and adhesion in whole blood aggregation. Despite attenuated platelet aggregation, SSCKO platelets at steady state show comparable expression of activated GPIIbIIIa relative to their AA and SS counterparts, indicating that the impaired aggregation at steady state is not due to defective integrin translocation. Since the Roche Multiplate Analyzer takes platelet adhesion into account in determining aggregation, we hypothesize that defective SSCKO whole blood aggregation at steady state is due to reduced cell adhesion. Finally, while silent infarcts were detected in all tissues, fibrin thrombi were not detected, a finding consistent with studies in the Berkeley model reporting minimal thrombi formation in tissues. In summary, we report that calpain-1 is required for platelet aggregation in steady state SCD but not following H/R treatment. This is the first study of H/R-induced platelet dysfunction in the Townes model, and raises the possibility of targeting calpain-1 as a treatment for platelet hyperactivity in SCD. [Display omitted] [Display omitted] DisclosuresJakubowski:Eli Lilly and Company: Employment, Equity Ownership. Frelinger:GL Synthesis: Research Funding; Celerion: Research Funding; Megakaryon: Research Funding; Bristol-Myers Squibb: Research Funding; Sysmex: Research Funding; Eisai: Research Funding; Baxalta: Research Funding; Pfizer: Research Funding; GE Global Research: Research Funding; NIH: Research Funding.

135 Cardiovascular Complications in Sickle Cell Disease

<h3>Introduction</h3> Sickle cell disease (SCD) is a genetic disorder affecting the production of haemoglobin (Hb). It has life-long consequences with multi-organ involvement including the cardiopulmonary circulation and an increased mortality. Although all SCD patients share the same genetic defect, the phenotype varies. This suggests other factors, unrelated to the Hb mutation, play an important role particularly in later decades. This retrospective study examined patient survival attending a specialist tertiary centre in the United Kingdom based on Tricuspid Regurgitant Jet Velocity (TRJ) measured by echocardiography and associated laboratory studies. <h3>Method</h3> A retrospective analysis of TRJ and diastolic function (lateral E/E’ ratio) assessed by echocardiography in 127 intensively treated steady-state SCD patients (mean age 40 +/- 12 years) screened for cardiovascular complications. Laboratory studies were also analysed. <h3>Results</h3> The clinical characteristics of patients in this study is summarised in Table 1. 15% of patients had a TRJ between 2.6–2.99 m/s and 9% with TRJ &gt;3.0 m/s. 11% of patients had diastolic dysfunction using lateral E/E’ ratio. When compared to patients with a TRJ &lt;2.5 m/s, the risk of death is 4 times higher in the 2.6–2.99 TRJ group and 24 times higher in the &gt; 3.0 TRJ group. See Figure 1. Lateral E/E’ ratio (beta = 0.88, p = 0.015), left atrial area (beta = 0.38, p &lt; 0.001) and microalbuminuria (beta = 0.10, p = 0.05) were independently associated with TRJ. <h3>Conclusion</h3> Mortality in SCD patients with raised TRJ remains high despite intensive therapy, such as exchange transfusion, directed at correcting Hb. Screening with echocardiography remains an important tool in identifying high-risk patients. Additional therapies directed at the premature ageing of the cardiopulmonary circulation are urgently needed.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea.

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Attenuating a sickle cell crisis with annexin V

A sickle cell crisis is a painful and dangerous condition that defies effective treatment but fortunately it usually terminates spontaneously and patients spend far more time crisis free than in its painful throes. This suggests that an unstable physiologic balance exists between steady state sickle cell disease (SCD) and the crisis state and if this is so a therapeutic nudge during a crisis may help to terminate it. Annexin V may be able to provide this push. The phosphatidylserine (PS) molecules normally appear on the surface of senescent erythrocytes where they are recognized by macrophages and rapidly removed so that normally only about 1% are present in the circulation but in SCD 30–40% are prematurely senescent and their removal is delayed. The PS+ sickle erythrocytes remaining in the circulation adhere to the endothelium and their exposed PS acts as a platform for the initiation of the coagulation cascade that is responsible for clot propagation. Annexin V’s great affinity for PS allows it to bond to it forming a shield that blocks both of these actions suggesting that its therapeutic administration during a sickle crisis may be able to hasten its termination.

Vitamin A Status of Steady State Sickle Cell Anaemia Patients Compared to Normal Control in Maiduguri North Eastern Nigeria

Vitamin A Status of Steady State Sickle Cell Anaemia Patients Compared to Normal Control in Maiduguri North Eastern Nigeria

Cardiac Dimensions and Functional Parameters in Nigerian Children with Homozygous Sickle Cell Anaemia Using Echocardiography

Background: Sickle cell anaemia (SCA) is the most common and burdensome inherited disease affecting children in Africa. Nonetheless, there remains a paucity of literature from Nigeria about its effects on cardiac dimensions and functioning in children. Objective: To determine cardiac dimensions and left ventricular (LV) systolic function in children with SCA in steady state and compare with same in healthy haemoglobin type AA controls. Methodology: Case-controlled, cross-sectional study of 50 subjects aged ≤ 15 years and 50 age- and sexmatched controls to determine and compare aortic root diameter (Ao), left atrium diameters (LAD), LV dimensions and indices of LV function [fractional shortening (FS) and ejection fraction (EF)] using 2-D guided M-mode echocardiography. Body surface areas (BSA) and venous haematocrits were also assessed. Results: Subjects had significantly higher mean Ao and LAD than controls (p=0.020 and p<0.001 respectively). The LV dimension indices measured were all significantly higher in subjects, excepting LV-posterior wall diameter in diastole. Almost half (44.0%) of the subjects had LV hypertrophy and almost all (95.5%) were eccentric. Nevertheless, subjects and controls had similar systolic cardiac function [FS (%); 32.04 vs. 32.29, p=0.871 and EF (%); 59.94 vs. 60.86, p=0.326] and these showed no correlation with age, BSA or haematocrit in both study groups. Conclusion: Nigerian children with SCA have deviations in cardiac dimensions with enlargements in various echocardiographic indices ranging from 1.1 to 1.4 times those of normal children. Yet, these children retain relatively normal systolic cardiac function.

Tissue factor-positive monocytes in children with sickle cell disease: relation to vaso-occlusive crisis

Background Hemostatic abnormalities are well documented in sickle cell disease (SCD). Nevertheless, whether these perturbations could contribute toward sickle vasculopathy is still not clear. Aim To evaluate tissue factor (TF) expression (CD142) on monocytes in children with SCD and correlate the results with the clinical state and some inflammatory and coagulation activation markers. Patients and methods This study included 24 children with SCD in steady state, 24 in painful crisis, and 20 healthy age-matched and sex-matched children as controls. The relevant data including the pain rate were retrieved from patients' files. For each participant, complete blood count, prothrombin time (PT%), activated partial thromboplastin time (aPTT), fibrinogen, d-dimer, thrombin-antithrombin complex, and quantitative C-reactive protein were assayed. TF expression on monocytes was analyzed by flow cytometry. Results TF-positive monocytes were significantly higher in both patient groups compared with the controls, being higher in patients in painful crisis (2.06 ± 0.64, 8.01 ± 1.53, and 13.5 ± 4.3 for the controls, steady-state group, and the painful crisis group, respectively, P Conclusion Collectively, these results confirm the prognostic significance of evaluation of TF-positive monocytes in SCD children.

Disease Severity Scores and Haemogram Parameters in Nigerian Sickle Cell Disease Patients

Background: Sickle cell disease (SCD) is associated with high mortality in Nigeria and the rest of sub-Saharan Africa; there is need to find easily available parameters that could predict disease severity and influence therapy. Objective: To evaluate the haemogram of a population of SCD patients and correlate these with objective scores of disease severity. Methods: Sixty (60) asymptomatic steady state (ASS) SCD patients in our clinic were randomly selected and interviewed with a questionnaire. Their haemogram was done using a 17 parameter, 3-part white cell differential, auto-analyser (KX 21N, Sysmex corporation, Chuo ku, Kobe, Japan) and objective severity scores calculated using a modification of the method proposed by Anyeagbu et al. Statistical analysis of data was done using Statistical Package for Social Sciences software, version 20 (SPSS Inc., IL, Chicago, USA), with significance assigned to p values less than 0.05. Result: Of the 60 subjects assessed, severity scores were calculated for 49: 11 (22.4%), 31 (63.3%) and 7 (14.3%) met the criteria for mild, moderate and severe disease respectively. The haemogram parameters that were significantly correlated with disease severity were mean corpuscular haemoglobin concentration (MCHC), and white blood cell count (WBC), p=0.014, and 0.001 respectively. Haemoglobin concentration (Hb) and packed cell volume (PCV) were negatively correlated with disease severity (p=0.001). Conclusion: In addition to already known haemogram parameters that affect SCD severity (such as WBC, Hb concentration, and PCV) MCHC also does same and can be manipulated by drugs and other kinds of therapy to ameliorate severity in patients.

Elevated levels of pro-coagulant microvesicles in children in-steady state sickle cell disease

Introduction: Sickle cell disease (SCD) is an inherited genetic disorder characterized by various complications, including thrombosis. Increased levels of circulating microvesicles (MVs) and tissue factor (TF)- bearing MVs have been reported in SCD. Objectives: The present study compares the levels of circulating MVs and TF- bearing MVs in steady state SCD children with age- and gender- matched healthy controls using an indirect ELISA. Citrated whole blood was collected from 54 SCD patients homozygous for sickle haemoglobin (HbSS) (aged from 2 to 12 years-old) and 34 healthy controls. Results: SCD patients showed significantly higher levels of MVs in their plasma as compared to the controls (P = 0.0095). Although the TF activity on MVs was low in both groups, there was a significant difference between them P <0.05). A strong correlation between the level of MVs and TF-MVs in the patient group was also noted. Conclusion: This suggests their involvement in the hypercoagulable state in the study group of patients. Further studies are recommended to elucidate the functional activity of MVs and TF-MVs, as well as the size and origin of MVs in the plasma.

Cytokines Polymorphisms and Relationship with Cytokine Expression in Sickle Cell Disease

Sickle cell disease (SCD) is a chronic inflammatory condition, even in steady state, as indicated by elevated levels of inflammatory cytokines and increased Th17 responses, compared with healthy controls. These inflammatory pathways may be directly regulated by genetic polymorphisms and could be associated to different outcomes of the disease. High levels of a number of different circulating cytokines were found in SCD patients in several studies, and changes in the cytokine balance in SCD patients are an important risk factor for the occurrence of clinical events. Moreover, inter-patient variations in cytokine levels could be attributed to gene polymorphisms. To investigate cytokine polymorphisms and their association with cytokines expression we evaluated the IL4 intron3 VNTR (genotypes 1.1, 1.2, 2.2, 2.3), IL4T590C/T, IL6174G/C and TNFA308A/G polymorphisms and their correlation with TGFB, IL-4, IL-6 and IL-10 expression in steady-state SCD patients.Methods. Fourth-nine patients (24 male and 25 female; 39.8 ± 9.59 years) with SCD and 28 (22 male and 6 female; 35.5 ± 10.2 years) healthy blood donors were evaluated. The polymorphisms were performed by PCR-RFLP analysis described as [individuals (genotype frequencies)] and the expression of TGFB, IL-4, IL-6 and IL-10 by q-PCR expressed as [median (max-min)].Results. A higher frequency of 1.2, 2.2 and 2.3 genotypes was found in SCD patients compared with normal controls [34(0.69) vs 12 (0.44), P=0.03]; higher expression of IL-4 was found in the ones carrying the 1.1 genotype [0.31 (2.53-0.01) vs 0.05 (0.95-0.0), P=0.047] and although no differences were found in the IL4T590C/T, IL6174G/C and TNFA308A/G polymorphism frequencies, a significantly greater expression of TGFB, IL6 and IL10 was observed in the patients cohort compared to normal individuals [1.55 (9.02-0.0) vs 0.97 (5.46-0.0), P=0.019]; [0.18 (45.45-0.0) vs 0.0 (8.14-0.0), P=0.03] and [0.98 (22.84-0.0) vs 0.0 (9.95-0.0), P<0.001, respectively]. All the genotype frequencies are consistent with Hardy-Weinberg equilibrium.Conclusion: A unique distribution of IL-4 genotypes was observed in our cohort of patients and controls, probably related to the miscellaneous ethnic background of our population. The highest prevalence of the IL4intron3 polymorphism in sickle cell patients suggests a less secretory phenotype associated with increased expression of inflammatory cytokines. IL-4 plays an important role in tissue adhesion and inflammation, including induction of adhesion molecules on vascular endothelial cells and could be responsible for a more “inflammatory” phenotype. Despite the small number of patients enrolled, our study brings insights and new data regarding the deregulation in immune system affecting SCD patients and this information must be investigated in larger cohorts, and may help to better characterize individual variations in immune responses and new markers for disease morbidity. DisclosuresNo relevant conflicts of interest to declare.

Ultrasonic assessment of the prevalence of gall stones in sickle cell disease children seen at the University of Benin Teaching Hospital, Benin City, Nigeria

Background: Gallstone is a common problem in patients with sickle cell disease. Prevalence of this problem among sickle cell disease (SCD) children may vary with age, and geographic location. Studies on gallstoneprevalence in SCD children are scanty in the South-South zone of Nigeria.Aim: To determine by ultrasonography the prevalence of gallstones among steady state sickle cell disease children attending the Paediatric Outpatient Clinic of the University of Benin Teaching Hospital, Benin-City, south southNigeria.Methodology: The study was a descriptive cross sectional study. The prevalence of gall stones was studied prospectively on 101 sickle cell disease patients who attended the Paediatric Outpatient clinic of the University of Benin Teaching Hospital by means of ultrasonography. They were aged 1-18 years. All except three patients were Haemoglobin (Hb) SSwhile the remaining three were HbSC. Statistical analysis was done by means, percentages and tests of significance was done using students t- test.Results: Five of the 101 children studied had gallstones and one had biliary sludge. The prevalence of gallstone was 5%. All patients with gallstones were HbSS, none with HbSC had stone although only three were studied. Only one child was symptomatic for the presence of gallstones. One patient had biliary sludge. The male: female ratio was 4:1. Prevalenceincreased with age; 2.9% 4.5% and 9.1% in patients aged less than 5,6-10 and 11-18 years respectively. Body weight was significantly associated with the presence of gallstones.Conclusion: Gallstone was found in 5% of the SCD children aged 1- 18years. This value is low compared to those from Europe and North America. It however confirmed the relative low prevalence in the general population of Nigerians. The clinical significance of gallstones in these SCD children is that it may contribute significantly to the morbidity and mortality seen in these children.Recommendation: Routine abdominal scan is recommended for sickle cell disease children to detect gallstone whose presence can mimic abdominal crisis. The option of elective cholecystectomy with reduced morbidity compared to emergency cholecystectomy can thus be exploited.Key words: Sickle cell disease, gallstones, biliary, sludge, Benin-City, ultrasound.

Significant bacteriuria in children with sickle cell anaemia in a Nigerian tertiary hospital

Background: Urinary tract infections (UTI) in children with sickle cell anemia (SCA) may result in long term morbidity and mortality due to chronic renal dysfunction. Objectives: To evaluate the prevalence of significant bacteriuria among children with SCA and to determine their antimicrobial sensitivity patterns of isolates. Methods: Two hundred and seventy two children with SCA in steady state (n = 185) and in crises (n = 87) aged 6 months to 15 years had their urine samples screened for significant bacteriuria. The urine samples were collected aseptically and incubated aerobically at 37‹C for 24 hours. Children whose urine samples yielded .105cfu/ ml of bacteria on two consecutive cultures were regarded as having significant bacteriuria. The antimicrobial sensitivity pattern of isolates was determined. Results: Significant bacteriuria was detected in 22 (8.1%) of the 272 subjects, 156 boys (57.4%) and 116 (42.6%) girls. The prevalence of significant bacteriuria was higher among those in crisis, 18 (20.7%) than among those in steady than, 4 (2.2%) state: ƒO2 = 27.323, p = 0.001. The most common organism isolated was Escherichia coli, 11 (50.0%). The antibiotic with the best sensitivity was ceftriaxone. Most organisms were resistant to the commonly used ant ibiot ic s l ike cotrimoxazole, amoxicillin and ampicillin. Conclusion: The prevalence of significant bacteriuria was found to be higher in SCA subjects in crisis (20.7%) than among those in steady state (2.2%). The most prevalent urinary pathogens were sensitive to ceftriaxone but resistant to commonly used antibiotics. Keywords : Sickle Cell Anaemia, Children, Prevalence, Significant Bacteriuria

LC-MS/MS analysis of plasma polyunsaturated fatty acids in patients with homozygous sickle cell disease.

The aim of this study was to determine circulating omega-6, omega-3 polyunsaturated fatty acids and prostaglandin E2 (PGE2) levels in steady state sickle cell disease (SCD) patients. Blood was collected from healthy hemoglobin volunteers and steady state homozygous HbSS patients who had not received blood transfusions in the last 3months. Plasma levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were determined by an optimized multiple reaction monitoring method using ultrafast liquid chromatography coupled with tandem mass spectrometry. PGE2 was measured in serum samples by enzyme immunoassay. Plasma AA and DGLA were significantly increased while EPA and DHA were significantly decreased in SCD plasma compared to control. Serum PGE2 levels, AA/DHA and AA/EPA ratio was significantly higher in SCD patients when compared to control group. The significant increase in PGE2 levels, AA/EPA and AA/DHA ratio confirms the presence of a proinflammatory state in SCD patients.

Endothelial Activation by Platelets from Sickle Cell Anemia Patients

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD.

The influence of decreased levels of high density lipoprotein cholesterol on hematological indices in sickle cell disease patients

Background:Changes in lipoproteins levels in sickle cell disease (SCD) patients are well-known, but the physiological ramifications of the low levels observed have not been entirely resolved.Aim:The aim of this study is to evaluate the impact of decreased levels of high density lipoprotein cholesterol (HDL-c) on hematological indices in steady state SCD patients.Subjects and Methods:The study was conducted on 84 SCD patients on steady clinical state, 36 males and 48 females with a mean age 21 (6) years. All those who have had blood transfusion within 4 months, infection, chronic kidney disease, and inflammatory episodes were excluded. Full blood count, total cholesterol, HDL-c, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, and triglyceride were assayed. Hematological indices of SCD patients with decreased levels of HDL-c were compared with those with normal HDL-c levels.Results:The SCD patients with decreased levels of HDL-c presented with lower levels of hemoglobin (P < 0.01), hematocrit (P < 0.001), total leukocyte count (P = 0.02), red blood cell count (P < 0.01), absolute neutrophil count (P = 0.04), absolute monocyte count (P < 0.01), and triglyceride (P = 0.02). Of the 47 SCD with decreased levels of HDL-c, 82.9% (39/47) have had blood transfusion while 48.6% (18/37) out of 37 SCD with normal HDL-c have had blood transfusion.Conclusion:SCD patients with decreased levels of HDL-c had more severe anemia, higher leukocyte and platelet counts than those with normal HDL-c levels. The low HDL-c marker may assist in the prediction of adverse clinical events in these patients.

Biochemical and ultrasound assessment of the liver in sickle cell anaemia patients in Zaria, Nigeria

Introduction: Sickle cell anemia (SCA) is frequently associated with liver disease. The constant state of hemolysis due to reduced red cell survival; and multiple blood transfusions, places these patients at risk hepatic sinusoidal congestion, viral hepatitis, cholestasis, and hemosiderosis, all of which may contribute to the development of liver disease. Objective: The aim was to assess the pattern of biochemical liver function tests and their relationship with liver size in steady state adult SCA patients in Zaria, North-West, Nigeria. Materials and Methods: Seventy-one adult SCA patients in steady state and 20 hemoglobin AA controls were enrolled into the study. Liver function tests were carried out and liver spans assessed using B-mode ultrasound scan in all subjects. Results: The serum bilirubin, aspartate transaminase (AST) and AST/alanine transaminase (ALT) ratio were significantly higher in SCA patients than in controls, with P values of 0.04, 2. Hepatomegaly was present in 67/71 (94%) of the patients, and absent in the controls. The liver span was significantly greater in SCA patients (P Conclusion: Steady state SCA patients in Ahmadu Bello University Teaching Hospital, Zaria, have hepatomegaly associated with minimal elevation of ALT and ALP. There was no correlation between the liver span and the biochemical parameters. It is advisable that liver function tests and liver ultrasound scan be interpreted with caution in these patients.

Antioxidant status of pediatric sickle cell disease patients with bacteraemia or malaria

Objective: Oxidative stress plays an important role in the pathogenesis of sickle cell disease (SCD) and many other infectious diseases. Antioxidant levels are known to be reduced in SCD and in malaria; however, there is no information on the antioxidant status of SCD patients with concomitant malaria or bacteremia. The objective of the present study was to determine potential effect of acute malaria or invasive bacteremia on selected antioxidant levels in SCD patients. Methods: The levels of ascorbic acid (ASC) and reduced glutathione (GSH), and activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured in red blood cell pellets of SCD children (n = 70) with (i) culture-proven bacteremia (n = 23), (ii) parasitologically confirmed malaria (n = 26) or (iii) steady state (n = 21). The antioxidant levels of SCD children were compared with those of non-SCD children (hemoglobin genotype HbAA, n = 51) with (a) parasitologically confirmed malaria (n = 26) or (b) non-malarial acute febrile illness (n = 25). Selected clinical, demographic and hematological parameters were determined for all recruited children. Results: The concentrations of ASC and activity of GPx were significantly higher in the SCD steady state patients compared with the SCD malaria patients, SCD bacteremia patients or non-SCD malaria patients. The GSH level was significantly higher in the non-SCD groups, compared with the SCD bacteremia patients or the SCD steady state patients. The levels or activities of these antioxidants were not increasingly reduced in the SCD patients with bacteremia or malaria. Hematological indices were significantly different between the SCD and non-SCD groups, and between the SCD malaria or bacteremia groups compared with SCD steady state patients. Conclusion: Antioxidant levels in SCD subjects with acute infections were low, but the potential effect of malaria or bacteremia was inconsistent, probably reflecting a complex relationship that merits further studies.