Hb AA Research Articles

The Homozygous Hemoglobin EE Genotype and Chronic Inflammation Are Associated with High Serum Ferritin and Soluble Transferrin Receptor Concentrations among Women in Rural Cambodia

Background: Ferritin and soluble transferrin receptor (sTfR) concentrations are commonly used to assess iron deficiency (ID); however, they are influenced by multiple factors.Objectives: We assessed associations between numerous variables and both ferritin and sTfR concentrations in Cambodian women and compared ID prevalence through the use of study-generated correction factors (CFs) for ferritin with those from a published meta-analysis.Methods: Venous blood from 450 women (aged 18–45 y) was assessed for hemoglobin (Hb), ferritin, sTfR, retinol binding protein, folate, vitamin B-12, C-reactive protein, α-1 acid glycoprotein (AGP), and genetic Hb disorders. Linear regression was used to calculate geometric mean ratios (95% CIs) for ferritin and sTfR concentrations.Results: The variant Hb EE genotype was associated with 50% (14%, 96%) and 51% (37%, 66%) higher geometric mean ferritin and sTfR concentrations, respectively, than was the normal Hb AA genotype; a 1-g/L increase in AGP was associated with 99% (50%, 162%) and 48% (33%, 64%) higher concentrations in the same variables, respectively. ID prevalence in nonpregnant women (n = 420) was 2% (n = 9) with the use of ferritin <15 μg/L and 18% (n = 79) with the use of sTfR >8.3 mg/L as criteria. ID prevalence with the use of sTfR was higher in women with the Hb EE genotype (n = 17; 55%) than in those with the Hb AA genotype (n = 20; 10%); and in women with the Hb AA genotype and chronic inflammation (n = 10; 18%) than in that group of women without chronic inflammation (n = 10; 7%) (P < 0.05). No differences in ID prevalence were found with the use of ferritin between women with Hb EE and AA genotypes (P = 1.0) or by chronic inflammation status (P = 0.32). There were no differences in mean ferritin concentrations among all 450 women when study-generated CFs were compared with those from the meta-analysis (P = 0.87).Conclusions: Compared with sTfR, ferritin concentrations appear to reflect more accurately true ID in rural Cambodian women. The CFs from a published meta-analysis were appropriate for use in this population with a high prevalence of Hb disorders and inflammation.

Impact of sickle cell trait on the thrombotic risk associated with non-O blood groups in northern Nigeria.

The non-O blood group is an established risk factor for deep vein thrombosis (DVT), while controversy surrounds the role of sickle cell trait (SCT) as a risk factor for DVT. We hypothesised that if SCT is a risk factor for DVT, individuals with non-O blood groups and SCT (Hb AS) would have a higher risk of DVT than their counterparts with non-O blood groups and normal haemoglobin phenotype (Hb AA). We retrospectively analysed the prevalence of SCT and non-O blood groups among 148 DVT patients with control subjects in order to determine the role of SCT as a risk factor for DVT and its impact on the risk of DVT among patients with non-O blood groups. In comparison with control subjects, DVT patients had significantly higher prevalences of SCT (35.1% vs 27.7%, p=0.04) and non-O blood groups (68.9% vs 45.9%, p=0.02). The odds ratios for DVT due to SCT, non-O blood groups with normal Hb phenotype (Hb AA) and non-O blood groups with SCT (Hb AS) were 1.3, 2.4 and 3.5, respectively. These results suggest that SCT by itself is a weak risk factor for DVT but it has the potential of escalating the DVT risk among patients with non-O blood groups. The combined effects of elevated clotting factors (non-O group effect) and increased clotting factor activation (SCT effect) were responsible for the escalated DVT risk among patients with co-inheritance of non-O blood groups and SCT. Co-inheritance of SCT and non-O blood group is, therefore, an important mixed risk factor for DVT. This should be taken into account when assessing DVT risk profiles of patients in Africa and other parts of the world where the SCT is prevalent.

Prevalence of β(S)-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil.

The aim of this study was to determine the frequency of beta S-globin gene (βS globin) haplotypes and alpha thalassemia with 3.7 kb deletion (−α3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of βS globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The βSglobin haplotypes and −α3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the −α3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity.

Alemtuzumab/Low Dose Radiation Conditioning Regimen in Allogeneic Hematopoietic Stem Cell Transplantation for Adult Patients with Sickle Cell Disease (SCD)

Allogeneic hematopoietic stem cell transplantation (HSCT) can cure SCD but chemotherapy-based conditioning regimens are associated with substantial morbidity and mortality, particularly in adult patients. A chemotherapy-free, non-myeloablative HSCT with alemtuzumab 1mg/kg divided over 5 days followed by total body irradiation (TBI) 300cGy was recently developed at the National Institute of Health and obtained stable mixed chimerism and clinical cure in 90% of SCD patients receiving an HLA and ABO-matched, related HSCT. The same study was replicated in high risk SCD adult patients at the University of Illinois at Chicago.Thirteen patients aged 17 - 40 years were transplanted between November 2011 and June 2014. Indications included ≥3 vaso-occlusive crises (VOC) per year (n=7), stroke (n=4) and >1 episode of acute chest syndrome (ACS) requiring intensive care (n=2). Unmanipulated, G-CSF mobilized peripheral blood cells were collected from HLA-matched, related donors (MRD) with Hb AS (n=4) or Hb AA (n=9). Four patients had red blood cell (RBC) alloimmunization and 2/13 patients received a graft with major ABO incompatibility. The median number of CD34+ cells infused was 8.1 x 106 cells/kg. Of 13 patients, 10 had severe neutropenia for a median duration of 5 days (range: 1 - 14) and 3 patients had a platelet count < 50 x109/L requiring a platelet transfusion. All patients engrafted and are alive with a median follow-up of 337 days (range: 53 – 986). Cytokine levels involved in T-cell regulation (IL-5, IL-10, and IL-13) increased post-alemtuzumab compared to pre-HSCT levels while no differences were observed in IL-2, IL-4, IL-12 or TNF-α. One secondary graft failure occurred in a patient not compliant with sirolimus and no acute or chronic GVHD has been observed. The median donor chimerism at 1 and 6 months was 96% (range: 63 – 98%) and 84% (range: 41 – 96%), respectively, in blood mononuclear cells; and 48% (range: 20 – 97%) and 25% (range: 20 – 48%), respectively, in CD3+ T cells (Figure 1a). Median hemoglobin concentration increased from 8.4 g/dL (range: 6.6 – 12.4 g/dL) to 12.9 g/dL (range: 11.0 – 16.8 g/dL) and the median reticulocyte percentage decreased from 12.1% (range: 4.8 – 19.6%) to 1.9% (range: 1.3 – 6.2%). Changes in hemoglobin fractionation in HSCT recipients from Hb AA donors are shown in Figure 1b. No episodes of ACS or stroke have been observed following hospital discharge after HSCT. Two patients have been tapered off of sirolimus with stable chimerisms, laboratory values, and no repeat episodes of SCD-related complications.Non-myeloablative HSCT from MRD with alemtuzumab and TBI in SCD adult patients provides a clinical cure in the majority of patients with low toxicity and no transplant-related mortality. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Divergence from Hardy Weinberg Equilibrium in Newborn Screening Cohorts for Sickle Hemoglobin

Newborn screening (NBS) for hemoglobinopathies was first introduced in the 1970’s to prevent infectious complications of homozygous sickle cell disease (hemoglobin [Hb] SS). The frequency of HbSS in newborns can be estimated using carrier rates (heterogygotes), assuming Hardy Weinberg Equilibrium (HWE). The current study examined whether NBS cohorts matched expected HWE distribution, and possible reasons for divergence. For the purpose of this study, all variants other than HbS were considered to be HbA. A total of 82 studies (1956 to 2013) were retrieved, of which 7 were excluded (carrier parents selected or screening sickle test used). Distribution of Hb SS, AS and AA was examined in 75 studies, from the following regions: Sub Saharan Africa n=26, Brazil n=12, USA n=12, EU n=10; Middle East/North Africa n=7, Caribbean n=6, India n=1, Canada n=1. The following screening laboratory methods were used: cellulose acetate electrophoresis (n=22), isoelectric focusing (n=21), high performance liquid chromatography (n=16), filter paper or micro column (n=7), other (n=9). A total of16,377,450 newborns or infants were included. In mixed random models, relative risk (RR) of detecting HbSS compared to expected HWE distribution was 2.06 (95% confidence interval [CI] 1.86, 3.66), p<0.001. In cohorts where HbAS carrier rates was >= 15%, RR was 1.44 (95% CI 1.23, 1.70), p=0.005; if carrier rates were between >=5% and <15%, RR was 1.62 (95% CI 1.20, 2.19), p=0.004; if carrier rates were <5%, RR was 5.74 (95% CI 3.4, 9.78), p<0.001. Thirteen studies provided data by subgroup: place of origin, ethnicity or race. Affected individuals were more likely to cluster in studies with lower HbS gene frequency (figure 1). Conversely, HbSS RR observed compared to expected HWE distribution was highest in populations with lowest overall gene frequency (figure 2). Overall RR for detecting HbAS compared to expected was 0.96 (95% CI 95, 97) p<0.001. Controlling for gene frequency, the Caribbean region was least likely to deviate from the expected HWE distribution, compared to other regions (RR 1.05, 95% CI 1.01, 1.05, P=0.007); cellulose acetate electrophoresis based methods were most likely to deviate from expected HWE distribution, compared to other methods (RR 0.97, 95%CI 0.95, 0.100, p=0.038). Deviation from HWE is commonly observed in NBS cohorts. It is an expected finding when smaller populations (e.g.migration) carrying a gene, exist within a larger population less affected with the same gene. However, even when controlling for gene frequency, regional variations were noted. Reasons may include further variations in population homogeneity, non-random mating within smaller groups (consanguinity), variations in hemoglobin variant distribution (alpha, beta thalassemia, fetal hemoglobin expression). Lab methods may also affect agreement with HWE distribution, as correct classification of non-affected individuals may vary between methods (normal homozygotes, variants and carriers). Divergence from HWE, especially when observed HbSS occurrence is less than expected, or when population Hb S gene distribution is known, may serve as a rapid and inexpensive quality control measure. Further examination of possible reasons for HWE deviation and caution when estimating burden of disease using carrier rates derived from NBS cohorts appear warranted. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Sickle Hemoglobinopathies on Pregnancy-Related Venous Thromboembolism

The aim of the study is to examine the relationship between sickle cell trait (Hb AS) and other sickle hemoglobinopathies and the risk of thromboembolism during pregnancy or the puerperium. Retrospective cohort study of African American women receiving prenatal care from 1991 to 2006. Sickle cell status was ascertained by routine hemoglobin electrophoresis. Venous thromboembolism (VTE) was defined as one or more episodes of deep venous and/or pulmonary thromboembolism during pregnancy or the puerperium according to discharge diagnoses based on International Classification of Diseases, Ninth Revision codes. Among 22,140 women with hemoglobin (Hb) AA status, 20 women (0.09%) experienced pregnancy-related VTE compared with 3 women (0.15%) of 2,037 women with Hb AS; relative risk (RR) for the association with AS status = 1.6; 95% confidence interval (CI) 0.5 to 5.5. Of 103 women, 3 women (2.9%) with sickle cell disease conditions (Hb SS, Hb SC, or Hb S,beta-thalassemia) experienced thromboembolism. Compared with women with Hb AA status, the RR = 32.2, 95% CI 9.7 to 107. Sickle cell trait may be associated with a modest increase in VTE in the setting of pregnancy; sickle cell disease conditions are strongly associated with this rare but potentially fatal outcome.

Extracellular Hemin Auto-Amplification Intensifies Tissue Injury In Sickle Cell Disease

Acute organ failure is a major clinical concern in sickle cell disease (SCD). However, the mechanism responsible for this potentially lethal complication is poorly understood. We tested the hypothesis that extracellular hemin liberates an intracellular danger molecule that promotes acute organ failure in SCD. Transgenic homozygous SCD (SS), sickle-trait (AS) and normal human hemoglobin (Hb) AA mice were infused with purified hemin (35 µmoles/kg), which raised total plasma hemin by ∼0.45 mM (equivalent to 0.72 g/dl Hb) within 5 min in all three groups of mice. In agreement with our previous results, SS but not AA and AS mice (n= 6 for each genotype) developed cardiopulmonary depression at 30 min evident by reductions in oxygen saturation (99.88±0.23% to 92.1±1.3%, p<0.001), breath rate (175.4±20.6 to 77.36±2.25, p<0.001, breath per min), heart rate (574.5±22.7 to 361.9±23.25 beats per min, p<0.001) and pulse distension (512.8±18.7 to 238.8±17.6 µm, p<0.001), and ∼70% of these animals died within 2 hours. Markedly raised lung wet/dry weight ratio in SS mice that succumbed to hemin suggests that the cardiopulmonary depression was secondary to a severe pulmonary edema. To identify biological correlates for the acute adverse effects in the SS mice, cohorts of both sickle and control mice were challenged with the same dose of hemin, blood samples were drawn at baseline (i.e. time=0 min), and 5 and 30 min after the hemin infusion and analyzed for markers of oxidative stress, tissue damage, plasma scavengers and high mobility group box-1 (HMGB-1), a prototypical danger molecule. Plasma hemopexin decreased by ∼80% at 5 min compared to baseline values in all three groups of mice regardless of the Hb genotype. The catabolism of hemopexin was associated with clearance of ∼50% of the hemin infusion from the circulation of AS and AA mice at 30 min. Paradoxically, the plasma concentration of hemin in the SS mice during this same time interval increased by ∼0.2 mM (p<0.001, n=6). The magnitude of this increase was dependent on the dose of hemin administered exogenously. We discovered that the de novo hemin release in the SS mice was preceded by acute intravascular hemolysis (mean decrease in total Hb: ∼1.4 g/dl, p<0.001, n=9, mean increase in cell-free Hb: 1.0 g/dl, p=0.001, n=9), oxidation of oxyHbS to metHbS (mean increase: 12%, p<0.001, n=6) and persistence of metHbS. It is noteworthy that de novo hemin release did not occur in AS mice suggesting that this phenomenon is dominantly influenced by sickle erythrocytes and not by the presence of intracellular HbS per se. Auto-amplification of hemin may help to explain an observation made nearly fifty years ago that SCD patient plasma contains more hemin than the plasma of patients with more severe intravascular hemolysis involving normal adult Hb (e.g. paroxysmal nocturnal hemoglobinuria), who have higher plasma Hb. To determine whether this phenomenon is critical to the cardiopulmonary depression in the SS mice, recombinant human hemopexin was administered 5 min after the infusion to sequester the endogenous hemin release. In hemin challenged SS mice with respiratory distress, intravenous recombinant human hemopexin rapidly halted the decline in oxygen saturation and breath rate and averted inevitable respiratory failure. In conclusion, we have identified a phenomenon of extracellular hemin auto-amplification that appears to be unique to SCD, and may play a critical role in propagating tissue injury in this disorder. Factors that inhibit erythrocyte lysis and accelerate metHb reduction may help to limit extracellular hemin amplification and preserve organ function during episodes of acute exacerbations in SCD. Disclosures:No relevant conflicts of interest to declare.

Influence of βS allele in the lipid peroxidation and antioxidant capacity parameters

The oxidative process plays a fundamental role in the pathophysiology of sickle cell anemia (SCA), and population and environmental characteristics may influence redox balance. The aim of this study was to evaluate lipid peroxidation and antioxidant capacity in Brazilian Hb S carriers undergoing different therapies. Blood samples from 270 individuals were analyzed (Hb SS, n=68; Hb AS, n=53, and Hb AA, n=149). Hemoglobin genotypes were assessed through cytological, electrophoretic, chromatographic, and molecular methods. Plasma lipid peroxidation and antioxidant capacity were measured by spectrophotometric methods. Patients with SCA who used iron-chelating drugs combined with hydroxyurea, associated with regular transfusions, showed lower levels of TBARS (P≤0.05), higher levels of TEAC (P≤0.01), and lower TBARS/TEAC ratio (R=255.8). The redox profile of Hb AS subjects was not statistically different (P>0.05) from that of Hb AA subjects. The data suggest that oxidative stress is lower in the patients with SCA who received regular blood transfusions associated with the combined use of HU and iron chelators than the group received only HU. The redox system of the Hb AS carriers is compatible with the control group.

Human bulbar conjunctival hemodynamics in hemoglobin SS and SC disease

The known biophysical variations of hemoglobin (Hb) S and Hb C may result in hemodynamic differences between subjects with SS and SC disease. The purpose of this study was to measure and compare conjunctival hemodynamics between subjects with Hb SS and SC hemoglobinopathies. Image sequences of the conjunctival microcirculation were acquired in 9 healthy control subjects (Hb AA), 24 subjects with SC disease, and 18 subjects with SS disease, using a prototype imaging system. Diameter (D) and blood velocity (V) measurements were obtained in multiple venules of each subject. Data were categorized according to venule caliber by averaging V and D for venules with diameters less than (vessel size 1) or greater than (vessel size 2) 15 µm. V in vessel size 2 was significantly greater than V in vessel size 1 in the AA and SS groups (P ≥ 0.009), but not in the SC group (P = 0.1). V was significantly lower in the SC group as compared to the SS group (P = 0.03). In AA and SS groups, V correlated with D (P ≤ 0.005), but the correlation was not statistically significant in the SC group (P = 0.08). V was inversely correlated with hematocrit in the SS group for large vessels (P = 0.03); however, no significant correlation was found in the SC group (P ≥ 0.2). Quantitative assessment of conjunctival microvascular hemodynamics in SS and SC disease may advance understanding of sickle cell disease pathophysiology and thereby improve therapeutic interventions.

Plasma Membrane-Derived Vesicles in Sickle Cell Disease: A Possible Indicator of the Continuous Endothelial Stimulation and/or Injury to Blood Cells

Plasma membrane-derived vesicles (PMVs) are released into circulation in response to normal and stress/pathogenic conditions. They are of tremendous significance for the prediction, diagnosis, and observation of the therapeutic success of many diseases. Knowledge of their functional properties would contribute to a better understanding of the pathological mechanisms leading to various diseases in which their levels are raised. The objective of this study was to quantify and compare the levels of PMVs in sickle cell disease patients (Hb SS and Hb SC) with non-sickle cell (Hb AA) subjects. The comparison will help us research and understand the processes that lead to their constitutive release in sickle cell disease patients than in their normal counterparts. A total of one hundred and fifty (150) sickle cell disease patients (study group) and blood donors (control group) that consented to partake in the study were recruited. There were 82 males and 68 females. Fifty (50) each of Hb SS, Hb SC and Hb AA samples were obtained. Sodium metabisulphite (sickling) test, Haemoglobin electrophoresis and quantification of PMVs were carried out on all the samples. The sickle cell disease patients had elevated levels; SS (38.89 ± 0.73 × 104/ml; p= 0.01) and SC (32.62 ± 1.18 × 104/ml; p = 0.01) as against the control subjects (Hb AA) who had average PMVs of 11.28 ± 0.29 × 104/ml PFP (mean ± SEM). It was concluded that both SS and SC (study) samples showed an increased count of PMVs as compared to the AA (control) samples, suggesting persistent endothelial stimulation and/or injury of blood cells leading to continuous shedding of PMVs in sickle cell disease patients.

Association of Haemoglobin types with Age, Coat colour and Head Profile in Red Sokoto goats of Abuja Nigeria

This study was conducted to determine the distribution frequency and the association of haemoglobin types with head profile, coat colour, and sex in smallholder goats of Abuja, Nigeria. The sex, coat colour and head profile were assessed by visual appraisal. The coat colour was categorized as dark brown (DB), brown (BR) and black (BL), while the head profile was categorized into sub convex (SCX) and sub concave(SCC). Haemoglobin type was analyzed by Electrophoresis in which 3 haemoglobin genotypes were identified; AA,AB, BB in addition with foetal haemoglobin (AC) .The association of the haemoglobin type with head profile, coat colour and age of goats were determine using chi-square analysis procedure of SAS. The result revealed that majority of the goats had SCX head profile (67%) with DB coat colour (68.1%). The frequency of heterozygote Hb AB (43.6%) was higher than the homozygotes Hb AA (32%) and Hb BB (11.7%) as well as the foetal haemoglobin (12.8%). However, the Hb A alleles had higher preponderance than Hb B allele. The distribution of Hb-types was found to be independent of age, coat colour and head profile of the goats. However, the foetal Hb type AC was mainly observed in young goats that were within one year of age (83%) and non beyond 2 years of age. The head profile was strongly (P<0.01) associated with sex of the goats, but independent of the coat colour. The SCX head profile was more peculiar to females than the males, while the SCC head profile was associated with males than females.

A Simple, Rapid, Low-Cost Test for the Diagnosis of Sickle Cell Disease Using a Paper-Based Hemoglobin Solubility Assay

Abstract 245▪▪This icon denotes a clinically relevant abstract Introduction:Sickle cell disease (SCD) is the most common inherited blood disorder caused by a mutation in the beta-globin chain of the hemoglobin (Hb) molecule. The result of this mutation, Hb S, polymerizes when deoxygenated, giving the red blood cells a characteristic, sickled shape. This polymerization causes in vivo vaso-occlusion and disrupts the vascular endothelium causing multi-organ complications in affected individuals. Nearly 70% of all affected births occur in Africa, where conservative estimates of SCD prevalence suggest a 10.68/1000 rate at birth (compared to 0.49/1000 in the U.S.). Although SCD causes significant morbidity and premature mortality, most affected persons born in the U.S. are able to survive into adulthood (primarily due to the implementation of universal newborn screening). In contrast, most affected individuals born in low-income countries die before the age of 5 years due to lack of early intervention. Current technology for diagnosing SCD relies on hemoglobin electrophoresis, isoelectric focusing or high performance liquid chromatography. This type of testing remains prohibitively expensive to low-income countries, where SCD is most prevalent and newborn screening is unavailable. Thus, many affected individuals are not diagnosed before severe complications or death can occur. The urgent need to develop a low-cost diagnostic test for SCD has been recently recognized as a priority by the World Health Organization. In this study, we describe the implementation of a paper-based hemoglobin solubility assay as a simple, low-cost and rapid test for definitive diagnosis of SCD. Materials and Methods:Normal human venous blood (Hb AA) was collected and compared to blood from patients with SCD (Hb SS) and SCT (Hb AS). To perform the test, a 20uL droplet of whole blood mixed with the components of the Hb solubility assay (SickleDex™, Streck) was deposited onto a paper-fluidic device fabricated using a previously published method. Polymerized deoxy-Hb S remained in the center of the blood stain entrapped by the paper substrate, while other forms of Hb remained soluble and were transported laterally to the periphery of the stain by capillary action. The resulting blood stain was digitized with a portable scanner and analyzed automatically. The red color intensity profiles were normalized by the total area under the curve to account for the differences in hematocrit among subjects. The SCD index was defined as the normalized color intensity 5 mm from the center of the blood stain. Data were presented as results of individual experiments and as mean ± SD. The values of the SCD index for samples from each Hb genotype were compared using a paired two-tailed t-test. Results:The entanglement of polymerized deoxy-Hb S by the fibers of the paper substrate resulted in the formation of a dark red spot in the center of the blood stain, while the wicking of the soluble forms of Hb from the center produced a pink ring on the periphery of the stain. The patterns of the blood stains produced on paper by normal (Hb AA), SCT (Hb AS) and SCD (Hb SS) samples were significantly different (Fig. A). The tight clustering of the normalized color intensity profiles for different subjects with the same Hb genotype enabled the use of the SCD index as a quantitative metric for distinguishing between the three types of samples (Fig. B). The difference between the SCD index measured for Hb AA, Hb AS and Hb SS samples was highly significant, with p<0.001 (Fig. C). Unlike the conventional, commercially available Hb solubility tests (e.g. SickleDex), the paper-based Hb solubility assay can conclusively differentiate between SCT and SCD samples using the characteristic blood stain patterns produced by each sample on the paper substrate. [Display omitted] Conclusions:This proof-of-concept study demonstrated the feasibility of using the paper-based Hb solubility assay as a simple, low-cost, point-of-care diagnostic test for SCD. The ability to diagnose SCD quickly and inexpensively will be particularly useful for universal SCD screening in resource-limited settings, such as Africa. This test will also be very useful in the emergency room setting in high-income countries to enable healthcare professionals to objectively confirm suspected SCD at the bedside. Disclosures:No relevant conflicts of interest to declare.

Effects of regular physical activity on skeletal muscle structural, energetic, and microvascular properties in carriers of sickle cell trait

To assess the effects of regular physical activity on muscle functional characteristics of carriers of sickle cell trait (SCT), 39 untrained (U) and trained (T) hemoglobin (Hb)AA (CON) and SCT subjects (U-CON, n = 12; U-SCT, n = 8; T-CON, n = 10; and T-SCT, n = 9) performed a graded exercise and a time to exhaustion (T(ex)) test, and were subjected to a muscle biopsy. Maximal power, total work performed during T(ex), citrate synthase and cytochrome c oxidase (COX) activities, respiratory chain complexes I and IV content, and capillary density (CD), diameter (COD), and surface area (CSA) were upregulated by the same proportion in T-CON and T-SCT compared with their untrained counterparts. These proportionally similar differences imply that the observed discrepancies between U-SCT and U-CON remained in the trained subjects. Specifically, both CD and COX remained and tended to remain lower, and both COD and CSA remained and tended to remain higher in T-SCT than in T-CON. Besides, carriers of SCT displayed specific adaptations with regular physical activity: creatine kinase activity; complexes II, III, and V content; and type I fiber surface area and capillary tortuosity were lower or unchanged in T-SCT than in U-SCT. In summary, our results show that 1) carriers of SCT adapted almost similarly to CON to regular physical activity for most of the studied muscle characteristics, 2) oxidative potential remains altered in physically active carriers of SCT compared with HbAA counterparts, and 3) the specific remodeling of muscle microvascular network persists in the trained state.

Measurement of Th1, Th2 cytokines and white cell count in childhood haemoglobinopathies with uncomplicated malaria infection

Background/objective: The relative balance between Th1 and Th2 cytokines appears crucial in the outcome of infections. We assayed the levels of some proinflammatory Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and anti-inflammatory Th2 cytokines, IL-4 and IL-10 in homozygous haemoglobin (Hb) AA, heterozygous HbAS-genotyped and sickle cell (HbSS) individuals with uncomplicated Plasmodium falciparum malaria.Methods: Levels of Th1 and Th2 cytokines of 111 children aged 1–5 years with uncomplicated malaria and 89 healthy controls were determined by enzyme linked immunosorbent assay and haematological parameters were estimated using the automated Swelab counter (Boule Medical, Stockholm, Sweden).Results: Th1 and Th2 cytokine levels were significantly higher in HbAA and HbAS-genotyped patients compared to their respective healthy controls (P<0·05). IFN-gamma, IL-2, and IL-10 were significantly elevated in HbAA compared to HbAS and HbSS subjects (P<0·05). The mean haematological parameters (total white cell count and monocytes) of HbSS-infected children were significantly higher compared to those of HbAA and HbAS subjects (P<0·05); however, their mean packed cell volume was significantly lower compared to others (P<0·05).Conclusion: Our results showed a stronger Th1 cytokine response in HbAA than HbAS and HbSS individuals; this may suggest an immunocompetence of the HbAA individuals in early infection.

Iron store of pregnant women with hemoglobin SS and SC in Benin city

Background: Anemia in pregnancy is common and morbidity is increased in the presence of sickle cell disease. There has been conflicting argument concerning routine iron replacement. However few, studies in Nigeria have comprehensively evaluated the iron status of these women. Objective: The study was carried out to determine the iron stores status of pregnant women with hemoglobin (Hb) Sickle cell S or Sickle cell C using the serum assay. C-reactive protein (CRP) was also assayed to rule out the effect of inflammation / infection on the serum level of ferritin. Materials and Methods: This was a prospective longitudinal study conducted at the University of Benin Teaching Hospital (UBTH), Benin City, over a period of eight months (from April 2011 to December 2011). The patients for the study were identified using the booking investigation results in the antenatal clinic records. Only those clinically stable pregnant women, in a healthy state, participated in this study.The hematological indices, serum ferritin assay, and the CRP were determined in both test (Hb SS / SC) and control (Hb AA) pregnant women. The data obtained were fed into a personal computer (PC) and analyzed using the Statistical Package for Social Sciences (SPSS) software version 16 for Windows. Categorical data were expressed as percentages and compared using the Chi-squared test, whereas, numerical data were expressed as mean (SD) and compared using the Student’s t-test. The level of significance was set at P≤0.05. Results: A total of 23 Hb SS / SC pregnant women formed the test group. They were recruited for the study from the Sickle-Cell Center (attached to the Central Hospital, Benin City, the UBTH). The mean age, educational status, and the social class of both the test group and the control group were comparable. There was a significant difference in the mean hemoglobin concentration between the test group and control group, both at 16–20 weeks and 28 – 32 weeks of gestation (P<0.001), and the mean corpuscular volume (MCV) values at both gestational ages (GAs) (P=0.097 and 0.231, respectively). The values of the serum ferritin in the test group were also statistically and significantly higher than those of the control group (P<0.001 and P=0.009, respectively), at both GAs. Conclusion: This study demonstrated higher serum iron levels in Hb SS / SC pregnant women than in the controls (even after excluding those levels raised by the increased levels of CRP). In situations that may require iron supplementation, it may be reasonable to determine the iron status to ascertain the quantity that should be given.

Prevalence and Significance of Sickle Cell Trait in Nigerian Patients with Essential Hypertension

Dr Sagir G. Ahmed, Department of Haematology Aminu Kano Teaching Hospital PMB 3452, Kano Kano State, Nigeria. +2348034418015 drsagirahmed@yahoo.com Abstract lifestyle and nutritional culture of the Africans are Global epidemiology shows that hypertension becoming increasingly westernized over time and is severer, commoner and occurs at relatively younger might be partly responsible for rising incidence of nonage in the black Africans in comparison to the whites. communicable diseases, including hypertension, in Since sickle cell trait (SCT) was associated with Africa. This is consistent with the finding of higher hyperviscosity, we hypothesize that SCT is a possible prevalence of hypertension in urban populations in 1 risk factor for hypertension in blacks and it might be Nigeria . Moreover, the prevalence of adult partly responsible for the high prevalence and severity hypertension appears to be rising as previous studies of hypertension in blacks. Hence, we studied reported prevalence rates of about 12% in 1992, 20% in 1, 2, 3 haematological parameters and prevalence of SCT in 1999 and up to 36% in a more recent study in 2008 . hypertensive patients and evaluated the impact of SCT Global epidemiology had shown that hypertension is on response to anti-hypertensive therapy as seen in severer, commoner and occurs at relatively younger 4, 5, Aminu Kano Teaching Hospital, Nigeria. age in the black Africans than in the white Europeans 6 The hypertensive patients had WBC and . 9 9 platelet counts of 5.8 x10 /L and 225 x10 /L that did not Individuals with sickle cell trait (SCT) have significantly differ from the corresponding values of red cells with Hb AS genotype that contain about 40% 9 9 7 6.2 x10 /L and 235 x10 /L seen among the HbS and 60% HbA . The abundance of HbA prevents 7 normotensive controls (p>0.05), but the hypertensive sickling under physiological conditions . The patients had a haematocrit of 0.37L/L that was frequency of SCT is up to 20-25% in West Africa significantly lower than the value of 0.44L/L seen including Nigeria, and the frequency has reached among the normotensive controls (p 0.05). Hypertensive patients with SCT had black Africans with normal Hb AA genotype . mean systolic and diastolic pressures of 158 and 95 Haemorheological studies had revealed that systemic mm Hg that were significantly higher than the arterial blood pressure is generally influenced by corresponding values of 145 and 86 mm Hg seen in vascular peripheral resistance, which is in turn affected 12, 13 patients with Hb AA genotype (p<0.05). These by blood viscosity . We therefore hypothesize that results suggest that SCT was not a risk factor for SCT genotype is a possible risk factor for essential hypertension but it was associated with poor hypertension in blacks and it might be partly response to anti-hypertensive therapy in affected responsible for the high prevalence and severity of patients. Hence hypertensive patients with SCT hypertension in the black race. If our hypothesis is need closer monitoring during therapy. correct, we predicted that: 1 The prevalence of SCT among black patients

Comparison of oxidative stress and the frequency of polymorphisms in the HFE gene between hemoglobin S trait blood donors and sickle cell disease patients

It is well documented that Hb S and iron affect blood cells, and trigger oxidative processes and generation of free radicals with potential for lipid peroxidation. We evaluated the frequency of polymorphisms in the HFE gene in Hb AS blood donors and how these polymorphisms influenced lipid peroxidation and antioxidant capacity. Blood samples were collected from 211 Hb AS blood donors, 119 Hb AA blood donors as a control group, and 28 sickle cell disease patients (Hb SS). The H63D allele was found at a frequency of 10.5% in the Hb AS samples, and the C282Y allele frequency was 0.7%. In the control group, the frequencies of the H63D and C282Y alleles were 13.4 and 2.1%, respectively. In the sickle-cell disease patients, the H63D and the C282Y allele frequencies were 10.7 and 3.5%, respectively. The frequencies of the C282Y and H63D polymorphisms in Hb AS blood donors are similar to those reported for the Brazilian population. Serum malondialdehyde values, indicative of lipid peroxidation, were highest in sickle cell patients, independent of the polymorphisms in the HFE gene, with significant differences, showing the influence of Hb S allele in the levels of lipid peroxidation. However, the trolox equivalent antioxidant capacity average levels, indicative of the antioxidant capacity, were reduced with significant differences, indicating that in spite of a lipid peroxidation raise, this is not followed by the increased of the antioxidant capacity, leading to oxidative stress.

Acute Chest Syndrome In Transgenic Mouse Models of Sickle Cell Disease Triggered by Free Heme

AbstractAbstract 944Acute chest syndrome (ACS) is the leading cause of death among patients with sickle cell disease (SCD). It is a process of devastating acute lung injury that evolves from multiple exacerbating events including vaso-occlusive pain crises, infection and fat emboli. ACS results in pulmonary infiltration, hypoxemia, and occlusions in the pulmonary microcirculation. Hitherto, experimental models of ACS have been lacking, and molecular targets of therapy remain to be identified. Clinical studies indicate that most patients diagnosed with ACS hemolyse during the acute phase of the syndrome, which highlights a role for circulating heme/hemin in this process. Since the deleterious effects of hemin are defined by increased vascular permeability, we tested the hypothesis that acute elevation of circulating hemin would increase pulmonary microvascular leakage sufficiently to trigger ACS. Adult transgenic mice expressing exclusively human sickle hemoglobin (Hb SS), and control Hb AS and Hb AA mice were intravenously injected with hemin (70 micromoles/kg body weight), and cardiopulmonary function assessed in real-time using a mouse pulse oximeter. Arterial oxygen saturation (SpO2) in the SS mice reduced significantly (p = 0.02) to 84.1 ± 5.6 % from a normal baseline value of 98.6 ± 0.3 %, within 25 minutes of administration of i.v. hemin, while SpO2 in control AS and AA mice remained unchanged. Consistent with changes in cardiopulmonary function, all the SS mice (n=14) succumbed to hemin, within 2 hours, while all control AS and AA mice survived, and remained alive several weeks after the experiment (log-rank survival test, p= <0.0001). We obtained identical results for survival in experiments using the Berkeley mouse model of SCD (Sickle 0/6, hemizygote 5/6, p=0.003). Post-mortem findings of gross pulmonary infiltration, alveolar flooding and microvascular occlusions, in the lungs of SS and Berkeley sickle mice that succumbed to hemin was consistent with respiratory distress associated sudden death. Younger SS mice aged 5–6 weeks were more resistant to i.v. hemin, with a survival rate of 80% (12/15), recapitulating the age-dependant mortality in human ACS. As expected, i.v. hemin raised the total plasma heme concentration to the same level in all mice, regardless of genotype. However, the concentration of protein-free plasma heme (PFPH) was increased by 6-fold in SS compared to AS mice (p = 0.001, n=12). The inability of SS mice to effectively scavenge excess free heme was likely because of very low plasma concentrations of hemopexin (SS: 0.17 ± 0.06 mg/ml, AS: 0.71 ± 0.14 mg/ml, p=0.002, n=8). We found a 10-fold higher concentration of heme oxygenase-1 (HO-1) in the plasma of SS mice, compared to AS mice (p=0.006, n=12), however, this enhanced capacity to degrade circulating heme, failed to protect the SS mice. This study demonstrates that acute elevation of plasma hemin triggers ACS in SCD mice. Infusion of hemopexin may prevent ACS during episodes of hemolytic crises in SCD. Disclosures:Ofori-Acquah:Emory University : Patents & Royalties.

Profil lipidoprotéinique, isotopique et risque athérogène dans la drépanocytose en Côte d’Ivoire

The principal goal of this study was to assess the possible disturbances of lipids and lipoproteins in sickle cell disease and establish a relationship between painful crisis and atherogenic risks by the atherogenicity index in Ivoirian adults. We analysed serum plasma lipid and lipoprotein profiles of 126 subjects with sickle cell anemia, and 55 “Hb AA” healthy individuals. The lipid and lipoprotein parameters studied were total cholesterol, triglycerids, HDL, LDL, apoproteins A1 and B, electrophoresis of lipoproteins and haemoglobin. In painful crisis, levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, apoproteins A1 (apo A1) in sickle cell anemia patients were shown to be significantly lower and levels of triglycerides higher than that of control group and steady state. The electrophoresis profile showed a significant fall of α lipoproteins while β lipoproteins were significantly high in period of crisis. The atherogenicity index (total cholesterol/HDL ratio) was also significantly high, just as LDL/HDL ratio in period of crisis. The overview of these results might hypothesize a high relatively atherogenic risk during the sickle-cell anemia crisis. A special monitoring of the patients in crisis is also necessary in order to prevent the risk of cardiovascular diseases.

Neuropsychological Dysfunction and Neuroimaging Abnormalities in Neurologically Intact Adults With Sickle Cell Anemia

Sickle cell anemia (SCA) is a chronic illness causing progressive deterioration in quality of life. Brain dysfunction may be the most important and least studied problem affecting individuals with this disease. To measure neurocognitive dysfunction in neurologically asymptomatic adults with SCA vs healthy control individuals. Cross-sectional study comparing neuropsychological function and neuroimaging findings in neurologically asymptomatic adults with SCA and controls from 12 SCA centers, conducted between December 2004 and May 2008. Participants were patients with SCA (hemoglobin [Hb] SS and hemoglobin level < or = 10 mg/dL) aged 19 to 55 years and of African descent (n = 149) or community controls (Hb AA and normal hemoglobin level) (n = 47). Participants were stratified on age, sex, and education. The primary outcome measure was nonverbal function assessed by the Wechsler Adult Intelligence Scale, third edition (WAIS-III) Performance IQ Index. Secondary exploratory outcomes included performance on neurocognitive tests of executive function, memory, attention, and language and magnetic resonance imaging measurement of total intracranial and hippocampal volume, cortical gray and white matter, and lacunae. The mean WAIS-III Performance IQ score of patients with SCA was significantly lower than that of controls (adjusted mean, 86.69 for patients with SCA vs 95.19 for controls [mean difference, -5.50; 95% confidence interval {CI}, -9.55 to -1.44]; P = .008), with 33% performing more than 1 SD (<85) below the population mean. Among secondary measures, differences were observed in adjusted mean values for global cognitive function (full-scale IQ) (90.47 for patients with SCA vs 95.66 for controls [mean difference, -5.19; 95% CI, -9.24 to -1.13]; P = .01), working memory (90.75 vs 95.25 [mean difference, -4.50; 95% CI, -8.55 to -0.45]; P = .03), processing speed (86.50 vs 97.95 [mean difference, -11.46; 95% CI, -15.51 to -7.40]; P < .001), and measures of executive function. Anemia was associated with poorer neurocognitive function in older patients. No differences in total gray matter or hippocampal volume were observed. Lacunae were more frequent in patients with SCA but not independently related to neurocognitive function. Compared with healthy controls, adults with SCA had poorer cognitive performance, which was associated with anemia and age.