Hb AA Research Articles

Activating pyruvate kinase improves red blood cell integrity by reducing band 3 tyrosine phosphorylation

AbstractIn a phase 1 study (NCT04000165), we established proof of concept for activating pyruvate kinase (PK) in sickle cell disease (SCD) as a viable antisickling therapy. AG-348 (mitapivat), a PK activator, increased adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate levels while patients were on treatment, in line with the mechanism of the drug. We noted that the increased hemoglobin (Hb) persisted for 4 weeks after stopping AG-348 until the end of study (EOS). Here, we investigated the pathways modulated by activating PK that may contribute to the improved red blood cell (RBC) survival after AG-348 cessation. We evaluated frozen whole blood samples taken at multiple time points from patients in the phase 1 study, from which RBC ghosts were isolated and analyzed by western blotting for tyrosine phosphorylation of band 3 (Tyr-p-bd3), ankyrin-1, and intact (active) protein tyrosine phosphatase 1B (PTP1B) levels. We observed a significant dose-dependent decrease in mean Tyr-p-bd3 from baseline in the patients, accompanied by an increase in the levels of membrane-associated ankyrin-1 and intact PTP1B, all of which returned to near baseline by EOS. Because PTP1B is cleaved (inactivated) by intracellular Ca2+-dependent calpain, we next measured the effect of AG-348 on ATP production and calpain activity and the plasma membrane Ca2+ ATPase pump–mediated efflux kinetics in HbAA and HbSS erythrocytes. AG-348 treatment increased ATP levels, decreased calpain activity, and increased Ca2+ efflux. Altogether, our data indicate that ATP increase is a key mechanism underlying the increase in hemoglobin levels upon PK activation in SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens

Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.

Assessment of hypoxemia among young adults with sickle cell anaemia in steady state in southwestern Nigeria: a crosssectional study.

Hypoxia is a known feature of sickle cell anaemia (SCA) which results from chronic anaemia and recurrent vaso-occlusive crisis (VOC) which can cause tissue ischaemia that leads to an end organ damage. The hallmark of SCA is chronic anaemia and recurrent vaso-occlusive crisis. The aim of this study is to compare the oxygen saturation of sickle cell anaemic individuals with the normal haemoglobin type (Hb AA) control and also to determine the prevalence of hypoxemia among SCA. Two-hundred and twenty-two (136 Hb SS and 86 Hb AA) participated in the study. The mean ± SD of age (years), oxygen saturation (%) and pulse rate (bpm) of participants with sickle cell anaemia and Hb AA control were 21.85 ± 3.04 and 22.14 ± 3.18 (t = 0.701, p = 0.436), 95.21 ± 3.02 and 98.07 ± 0.81 (t=-8.598, p < 0.0001) and 77.10 ± 9.28 and 73.16 ± 8.52 (t = 3.173, p = 0.002) respectively. The prevalence of hypoxemia among SCA participants was 47.1%. Prevalence of hypoxemia in males with SCA was 60.9% while 39.1% of the females had hypoxemia.

Immune responses to P falciparum antibodies in symptomatic malaria patients with variant hemoglobin genotypes in Ghana

BackgroundHaemoglobin (Hb) variants such as sickle cell trait (SCT/HbAS) play a role in protecting against clinical malaria, but little is known about the development of immune responses against malaria parasite (Plasmodium falciparum surface protein 230 (Pfs230) and Plasmodium falciparum erythrocyte binding antigen 175 region-3 (PfEBA175-3R)) and vector (on the An. gambiae Salivary Gland Protein-6 peptide 1 (gSG6-P1)) antigens in individuals with variants Hb genotypes. This study assessed antibody (IgG) responses against malaria parasite, Pfs230 and PfEBA175-3R and vector, gSG6-P1 in febrile individuals with variant Hb genotypes.MethodsThe study was conducted on symptomatic malaria patients attending various healthcare facilities throughout Ghana. Microscopy and ELISA were used to determine the natural IgG antibody levels of gSG6-P1, PfEBA175-3R & Pfs230, and Capillarys 2 Flex Piercing was used for Hb variants determination.ResultsOf the 600 symptomatic malaria patients, 50.0% of the participants had malaria parasites by microscopy. The majority 79.0% (398/504) of the participants had Hb AA, followed by HbAS variant at 11.3% (57/504) and HbAC 6.7% (34/504). There were significantly (p < 0.0001) reduced levels of gSG6-P1 IgG in individuals with both HbAC and HbAS genotypes compared to the HbAA genotype. The levels of gSG6-P1 IgG were significantly (p < 0.0001) higher in HbAS compared to HbAC. Similarly, Pfs230 IgG and PfEBA-175-3R IgG distributions observed across the haemoglobin variants were significantly higher in HbAC relative to HbAS.ConclusionThe study has shown that haemoglobin variants significantly influence the pattern of anti-gSG6-P1, Pfs230, and PfEBA-175 IgG levels in malaria-endemic population. The HbAS genotype is suggested to confer protection against malaria infection. Reduced exposure to infection ultimately reduces the induction of antibodies targeted against P. falciparum antigens.

Povišena srednja zapremina eritrocita u anemiji srpastih ćelija - jedna priča, previše?

Abstract: Introduction: Sickle cell disease is a hereditary blood disorder characterized by defective hemoglobin. Red cell indices are proposed as potential tools for diagnosing and managing sickle cell disorders. Materials and Methods: This study aimed to assess the utility of red cell indices as screening tools for sickle cell anemia. One hundred consenting adults of both sexes participated. Haematological parameters, including packed cell volume, hemoglobin values, hemoglobin electrphoretic patterns, and red blood cell count, were examined. Mean cell volume (MCV), mean cell hemoglobin concentration (MCHC), and mean cell hemoglobin (MCH) were calculated. Data analysis was performed using GraphPad Prism Software Version 9, with statistical significance set at p &lt; 0.05 (95% confidence interval). Results: Haemoglobin values were significantly lower in Hb SS subjects (5.68 ± 1.7g/dl) compared to Hb AA (11.30 ± 1.5 g/dl) and Hb AS groups (11.03 ± 1.4 g/dl) (F = 32.279; p &lt; 0.00001). The pattern was consistent with PCV and RBC values. Among the red blood cell indices assessed, only MCV showed a significant elevation (95.7 ± 2.4 fl) in the HbSS group compared to other groups (F = 4.165; p = 0.0183). No statistically significant difference was observed in MCHC and MCH values between the three groups (F = 0.5373, p &gt; 0.586 for MCHC; F = 0.607, p &gt; 0.546 for MCH). The prevalence of haemoglobin variants was as follows: HbAA (77%), HbAS (19%), and HbSS (4%). Conclusion: This study highlights significant reductions in haemoglobin values in Hb SS subjects and a notable elevation in MCV values in the Hb SS blood group. Elevated MCV in sickle cell anemia, where red cells are typically microcytic, warrants further investigation for differential diagnosis.

Donor Hemoglobin Genotype Does Not Impact Outcomes Following Matched Related Donor Hematopoietic Cell Transplantation for Sickle Cell Disease: A STAR Study

Sickle cell disease (SCD) is an inherited hemoglobin (Hb) disorder which leads to significant morbidity and early mortality. Hematopoietic cell transplantation (HCT) is the only curative treatment with long-term outcome data, demonstrating excellent symptom-free survival particularly when a matched related donor (MRD) is available. Most MRD have sickle cell trait (Hb AS), but trait donor HCT is considered acceptable by the FDA and without negative impact. Further, initial gene addition and editing approaches for SCD have resulted in a trait-like status with benefit. Sickle trait, however, is associated with complications such as papillary necrosis, bacteriuria, splenic infarction, and exercise-induced death. The primary aim of this study was to compare long-term outcomes following MRD HCT for SCD based on donor Hb genotype, to objectively describe any impact on outcomes. Retrospective data was available for this analysis on 182 SCD patients ≥1 year post MRD HCT at 10 participating Sickle Transplant Advocacy and Research (STAR) Alliance centers. Summary statistics were presented as mean (standard deviation) for continuous variables and count (%) for categorical variables. Comparisons were made between Hb AA and Hb AS donor groups using both parametric and non-parametric tests for continuous variables and Chi-square test/exact test for categorical variables, with p-value &amp;lt;0.05 set as significant. Amongst 182 patients undergoing MRD HCT, 67% (n=122) had a Hb AS donor ( Table). In the overall cohort, median age at HCT was 8.7 (4.8) years and most patients had HbSS (n=174; 96%) with a severe clinical phenotype (n=97; 54%). HCT was performed using bone marrow (n=166; 92%) with myeloablative conditioning (n=156; 86%) in the majority. Patients with a severe disease phenotype and Hb AS donor were significantly more likely to have recurrent vaso-occlusive pain crises as an indication for HCT (p=0.033), otherwise there were no differences in baseline patient or HCT characteristics between cohorts. With mean post-HCT follow up of 4.5 (3.9) and 5.1 (4.1) years in Hb AA and Hb AS donor cohorts (p=0.145), no significant differences were seen in recipient outcomes including time to neutrophil and platelet engraftment, number of or time to last platelet transfusion, myeloid or unsorted % donor chimerism, number with acute (day 100) or chronic GVHD (1 year), or time to discontinue immunosuppression (data not shown). Disease recurrence and need for 2 nd HCT were rare in both cohorts (AA: 2 [3%] and 2 [3%]; AS: 3 [3%] and 4 [3%]; p=0.665 and 1.0, respectively). Two (3%) and 5 (4%) patients died in the AA and AS cohorts, respectively (p=1.0). Aside from a statistically (but not clinically) significant difference in Hb at day 30 (p=0.044), no difference was seen in longitudinal Hb or markers of hemolysis (LDH and ARC; Figure). No significant difference was seen in median number of RBC units transfused (AA: 4.0 [2.0-6.0]; AS: 3.0 [2.0-6.0]; p=0.454) or days to last transfusion (AA: 23.0 [14.0-77.0]; AS: 22.0 [13.0-75.0]; p=0.86). SCD complications were rare post-HCT, with no difference seen between cohorts in number (%) with VOC (AA: 1 [2%; had 2 separate VOC events]; AS: 3 [3%]; p=1.0), ACS (none reported), chronic pain (AA: 0 [0%]; AS: 1 [&amp;lt;1%]; p=1.0), or other SCD-related complications (AA: 2 [3%]; AS: 9 [7%; had 11 separate other events]; p=0.456). Stroke occurred in a significantly higher proportion with an AA versus AS donor (5 [8%] and 2 [2%], respectively; p=0.04). No significant differences were seen in longitudinal (1 and 2 years and at last follow up) post-HCT cardiac (EF, SF, TRJ velocity), pulmonary (FEV1, FVC, DLCO), or renal (GFR) function between cohorts (data not shown). Our results indicate that in MRD HCT for SCD, AS and AA donors yield comparable long-term outcomes. In this retrospective and registry-based study design, detailed analyses did not show differences between the two cohorts at baseline or at any timepoint post-HCT. While there were some outcome differences between the cohorts, they were either not clinically significant or did not have a scientific rationale, and thus were likely due to chance alone (denoted by bolding in Table). Given the young age of our cohort at HCT and thus a smaller proportion with SCD-related organ dysfunction, our results may not be applicable to an older cohort with significant organ dysfunction which will require a separate analysis.

Association of Cerebral Hemodynamics and Anemia on Processing Speed in Adults with Sickle Cell Disease.

Compared to healthy controls, adult patients with Sickle Cell Disease (SCD) are anemic, and therefore have higher cardiac output and Cerebral Blood Flow (CBF) to maintain brain oxygenation. They also demonstrate comparatively more cognitive deficits due to either overt strokes or silent cerebral ischemia. However, there are few correlative studies between CBF and cognitive deficits, specifically processing speed in SCD. Such studies are important to develop biomarkers of central brain processing and ischemia for diagnosis, prognosis, and evaluating the effectiveness of potential interventions. This pilot cross-sectional study tested the hypotheses that adults with SCD and elevated CBF demonstrate lower central brain processing speed than controls on average and that CBF is inversely correlated with processing speed. We conducted a pilot cross-sectional study to assess the relation-ships between CBF, central brain processing speed, and hemoglobin levels in asymptomatic adults with SCD and controls from an urban academic medical center. MRI acquisitions at 3T consisted of 2D phase-contrast quantitative arteriograms (Qflow) of the bilateral internal carotid and vertebral arteries and 3D pseudo-continuous arterial spin labeling (pCASL) of the brain. Participants were patients with SCD (hemoglobin [Hb]SS, [Hb] SBetaThal°, or [Hb]SC) aged 22-52 years of African American descent (N=7) or community controls (Hb AA) (n=3). Processing speed was assessed as an in-direct functional marker of ischemia using a recommended test from the NIH Toolbox for Assessment of Neurological and Behavioral Function, the Pattern Comparison Processing Speed Test. t-tests were used to compare means of CBF, hemoglobin, and cognition between SCD patients and healthy controls. Among SCD patients only multivariate correla-tions were used to evaluate relationships between brain perfusion in specific brain regions vs. processing speed and CBF. The significance level was set at p≤0.05. Adults with SCD reported higher CBF compared to healthy con-trols (72.15±28.90 vs. 47.23±12.30 ml/min/100g, p=0.04), and lower hemoglobin concentration (8.64±2.33 vs. 13.33±0.58, p=0.001). Heart rate in SCD patients was higher than in controls (86.29±1.37 vs. 74.00±2.10, p=0.04). Patients with SCD demonstrated lower processing speed (96.14±21.04 vs.123±13.74, p=0.02) than controls. Among adult patients with SCD, perfusion in specific regions of the brain showed an inverse relationship with processing speed, as did whole-brain CBF (p=0.0325). These findings, although from a small sample, lend a degree of validity to the claim that processing speed is slower in people with SCD than in controls and that CBF is significantly higher in SCD patients com-pared to controls. The results also lend credence to the finding that the degree of processing speed deficiencies among adults with SCD is correlated with the degree of elevated CBF, which is known to correspond with the degree of anemia associated with SCD.

Haemoglobin Haplotypes and Probable Allelic Frequencies of natives of Okolobiri Community, Nigeria: A population genetic study

Introduction: Sickle cell disease is an autosomal recessive disorder in which patients inherit one mutated copy of the β-globin gene from each parent. This inheritance results to sickle-shaped red blood cell and associated fragility which results in the destruction of the patients red blood cells. This project was carried out as a population genetic study to determine the frequencies of the haemoglobin haplotypes (A and S) and allelic frequencies with a view to predicting the dynamics of the abnormal genes using a typical African community as a case study. Materials and Methods: Two hundred and twenty-eight (228) subjects constituted the study population of which one hundred and sixty four (164) were males (28%). Haemoglobin electrophoresis membrane at a pH 8.6 was used to establish the haemoglobin electrophoretic pattern of the study population. Results: The haemoglobin electrophoretic pattern of the study participants were as follows: Hb AA (60.5%), Hb AS (25.5%) and HbSS (14%). The distribution among males were; HbAA (62.2%), HbAS (29.3%) and HbSS (8.5%). and females: Hb AA (56.3%), HbAS (15 .6%), HbSS (28.1%). The HbA haplotype frequency was 0.732 while the HbS was 0.268. the allelic frequencies obtained by Hardy-Weinberg calculation were as follows. HbAA (0.536), HbAS (0.392) and Hb SS (0.072). The χ2 value of 29.475 obtained in this study was higher than the 3.84 at 5% significant level, hence, the null hypothesis that the population is not in Hardy-Weinberg equilibrium was accepted.. Conclusion: The study revealed high homozygous HbSS inheritance among females (28.1%) meaning that females are more affected than the males. The frequency of HbAS is expected to increase as this population is not in Hardy-Weinberg equilibrium. More awareness and sensitization is needed in our communities in order to reduce the prevalence of sickle cell anaemia in our African society.

An Estimation of Haemoglobin Variants Among Patients Attending A Tertiary Hospital In South West Nigeria

Background: Globally, hemoglobinopathies, which are genetic diseases of hemoglobin production, are a major cause of morbidity and mortality. African communities make up a sizable portion of the population that is susceptible to a variety of erythrocyte genetic and homological illnesses. Aim: The aim of this study is to investigate the prevalence and spectrum of hemoglobin variants among patients attending a tertiary hospital in South West Nigeria. Materials and Methods: The study research was conducted at the University of Medical Sciences Teaching Hospital, Ondo State Southwest Nigeria. Respondents were recruited from the hospital department clinics and tested at Department of Haematology, Faculty of basic medical science. Blood will be drawn via venipuncture into tubes that had been treated with the anticoagulant ethylene diamine tetracetic acid (EDTA) Results: The study show the frequency and gender distribution of different forms of haemoglobin (Hb) among the subjects; Hb AA 264; HbAS 60; HbAC 6; HbSC 8 and HbSS 12. Of the 350 subjects studied, 106 were males, while 244 were females. Among the male subjects, 85 were HbAA, 12 were HbAS; 2 were HbAC; 3 were HbSC and 4 were HbSS. Among the female subjects, 179 were HbAA, 48 were HbAS; 4 were HbAC; 5 were HbSC and 8 were HbSS. The study shows the frequency of the fetal hemoglobin among the Hb SS patients in the study based on age range. 50% of the participants with fetal hemoglobin were between 10-15 years, 25% were between 16-20 years, 8.3% were between 21-25 years, 8.4% were between 31-35 years and 8.3 % were between 36-40 years Conclusion: The study showed increased prevalence of various hemoglobin variants. Awareness should be created at religious centers, schools on the importance of genotype testing. As a preventative precaution. We advise the implementation of mutation identification and carrier screening.

Pattern of Hb genotype in surgical patients at a tertiary hospital in Southern, Nigeria

Background: Haemoglobin (Hb) genotype is not a routine test in adult pre-operative investigations in majority of clinical settings, though in the obstetric and gynaecological patients already have their Hb genotype as a part of the routine antenatal investigations. However, in paediatric work up for surgical procedures Hb genotype is a routine investigation. This is due to sickle cell endemicity in our locality. Aim: To determine the pattern of HB Genotype in surgical patients at the Rivers State University Teaching Hospital (RSUTH). Method: This was a one-year retrospective study of surgical patients (Surgery and Obstetrics/Gynaecology departments) of the RSUTH. The patients comprised of all the consecutive cases of the surgeries in these departments for the period under review. Ethical clearance was obtained from ethical committee of the Rivers State Hospital Management Board. Structured profoma was used to extract information from patients’ case notes and analyzed using SPSS version 25. Result: There were a total of 370 subjects that were recruited for the study under review. There were 146 (39.5%) males and 224 (60.5%) females. The mean age was 31 years. The age range was 22 years to 56 years. One hundred and ninety four (52.4%) were obstetrics and gynaecological surgeries while 176 (47.6 %) were non-gynaecological surgeries. The commonest indication for surgery was caesarean representing 126 (34.1%) of the subject. The most common Hb genotype was AA 246 (66.5%)of which females contributed to 148 (40%) and males 98(26.5%)had Hb AA while 124 (33.5%)of the subjects were Hb AS. Females that were Hb As were 74 (20%) while males were 50 (13.5%). There were no subjects that were Hb SS. Conclusion: This study showed the most prevalent Hb Genotype as AA (66.5%) in surgical patients at the Rivers State University Teaching Hospital (RSUTH), while 33.5% had Hb AS genotype. None of these subjects had sickle cell (HbSS) homozygous trait

Complement Drives Acute Chest Syndrome in a Model of Sickle Cell Disease

Background: Despite being the first genetic disease described, sickle cell disease (SCD) continues to afflict millions of individuals worldwide. Patients with SCD can experience severe complications, including vasoocclusive crisis (VOC), which can progress to acute chest syndrome (ACS), a form of acute lung injury. While VOC can cause significant morbidity, ACS is the leading cause of mortality in patients with SCD. Despite the devastating consequences of ACS, no interventions exist that directly treat this complication. As a result, when patients present with ACS, options often remain limited to supportive care. The lack of optimal treatment options for patients with SCD experiencing ACS directly contributes to their morbidity and mortality. Using a combined approach of prospective observational studies in patients with SCD that experienced ACS and a novel preclinical model specifically designed to define the role of complement in SCD pathophysiology, we tested the hypothesis that complement plays a central role in the pathophysiology of ACS. Methods: Samples from patients with SCD experiencing ACS were compared to baseline measurements for the same patient obtained at outpatient follow up 4-6 weeks later. ELISA based assays were used to measure Ba, Bb, C3a, C5a, C5b-9 (membrane attack complex (MAC)) in each sample. SCD mice (HbSS) and control mice (HbAA) were injected with 7.5 units of cobra venom factor (CVF), a potent complement activator, followed by evaluation of hematocrit (Hct) measured by a Sysmex veterinary hematology analyzer, free heme by calorimetric assay, and complement deposition on red blood cells (RBCs) by flow cytometry or pulmonary endothelial cells by confocal analysis. Respiratory rate and O2 saturation were measured using a MouseOx pulse oximeter. HbSS or HbAA mice were crossed with complement component 3 (C3) knock out (KO) mice to generate HbSS x C3 KO and HbAA x C3 KO mice. Hemin (70 to 210 mmol/kg concentration) or CVF (7.5 units/mouse) were injected into HbSS, HbAA, HbSS x C3 KO or HbAA x C3 KO followed by evaluation of hemolysis, C3 deposition and pulmonary function. A one-way ANOVA with a Tukey's post hoc with a p value <0.05 was considered significant. Results: During ACS in patients, significant elevations in Ba and Bb, complement activation products unique to the alternative pathway, and downstream activation of complement, including C3a, C5a and MAC, were observed (p<0.0001). Changes in complement activation were accompanied by a drop in hemoglobin during ACS when compared to baseline (p<0.001), suggesting that complement activation results in hemolysis. To formally test this, the impact of complement activation in a preclinical model of SCD was defined. Injection of CVF or hemin resulted in rapid C3 deposition on RBCs and pulmonary endothelium in HbSS, but not HbAA, recipients. In addition, CVF or hemin injection was accompanied by increased hemolysis and ACS manifested by increased respiratory rate, drop in oxygen saturation and death within 2 hours in the majority of recipients (p<0.001). In contrast, control Hb AA mice were unaffected by CVF or hemin injection. Given the role of free heme in the development of ACS, we next defined the role of complement in hemin-induced lung injury. To accomplish this, SCD mice were crossed with C3 KO mice to generate HbSS x C3 KO. HbSS x C3 KO mice displayed a mild increase in Hb values at baseline (p<0.01), suggesting a role of C3 even in steady-state SCD-related hemolysis. Equally important, injection of HbSS x C3 KO mice with CVF or hemin failed to result in the same levels of C3 deposition, hemolysis, compromised pulmonary function, or mortality (p<0.001) as HbSS recipients. Discussion: These results demonstrate a critical role for C3 in the development of acute lung injury in SCD. The sensitivity of HbSS RBCs to complement-induced hemolysis and C3 deposition observed following hemin injection in SCD recipients suggests that C3 plays a key role in decreased hematocrits and pulmonary injury that accompanies ACS. As treatment of ACS largely relies on supportive care, these results suggest that approaches aimed at targeting complement activation may represent a useful prophylactic or treatment strategy for ACS. In doing so, these results hold promise in providing a more direct approach at reducing one of the most severe complications of patients with SCD.

Potential of point of care tests for newborn screening for sickle cell disease: Evaluation of HemotypeSC™ and sickle SCAN® in Tanzania.

Sickle cell disease (SCD) is an important cause of <5 mortality. In Tanzania, it is estimated up to 11 000 children are born with SCD annually, making this the fifth country with the highest SCD annual births worldwide. The biggest challenge of expanding the service of newborn screening for SCD as the national health intervention in Tanzania is due to the high cost of the currently used assays and lack of rapid screening methods. Therefore, in this study, we assessed the diagnostic accuracy of point-of-care tests for SCD diagnosis in newborns. To evaluate the sensitivity and specificity of HemotypeSC™ and sickle SCAN® in diagnosing SCD in newborns. Diagnostic accuracy of HemotypeSC™ and sickle SCAN® were evaluated in comparison to isoelectric focusing as a confirmatory method. A total of 706 newborns were enrolled in the study. The sensitivity and specificity of HemotypeSC in detecting Hb SS, Hb AS and Hb AA phonotypes was 100%. The sensitivity and specificity of sickle SCAN® in detecting Hb SS, Hb AS and Hb AA phenotypes were 100%, 97% and 100% respectively. Both POC tests displayed high accuracy in detecting SCD, we believe the introduction of either of these tests in health facilities will help in the early detection and management of SCD. In addition, the margin of cost per test is relatively affordable (1.4$ per test for HemotypeSC™ and 4.75$ for sickle SCAN®).

Apohemoglobin‐haptoglobin (Apohb‐Hp) Complex as a Vasculo‐protective Agent in Hypoxia Induced Vaso‐occlusion in Sickle Cell Disease

Sickle Cell Disease is associated with episodes of vaso‐occlusive crises (VOCs) caused by hemoglobin S (HbS) polymerization and resultant microvascular endothelial dysfunction due to hemolysis, inflammation, defects in nitric oxide metabolism and oxidative stress. In the acute setting, care for VOC involves supportive care including hydration and aggressive pain management. There are no specific therapies known to alter the course of the disease during an acute VOC. Apohemoglobin‐haptoglobin (Apohb‐Hp) complex is a two‐protein formulation that scavenges free hemoglobin (Hb) as well as free heme and has been shown to have a role in alleviating vasoconstriction and hypertension induced by cell free Hb1. The objective of this study was to evaluate microcirculatory effects of Apohb‐Hp during hypoxia induced vaso occlusion in sickle cell mice.We used intravital microscopy on the dorsal skin fold to observe the microcirculatory effects of Apo Hb‐Hp, haptoglobin (Hp), vehicle control in HbSS‐Townes mice (6‐8 week and ~25 g) and Hb AA mice. Each group consisted of 11 mice, they were initially kept in normal conditions (21% O2) for an hour followed by a hypoxic state (7% O2) for another hour and finally brought back to normal conditions (21% O2) for an hour before being sacrificed. In some cases, some mice were followed for 72 hours before being sacrificed. ApoHb‐Hp and Hp were injected intravenously at a concentration of 59 mg/mL and 86 mg/mL respectively at a single dosing quantity of 150 mg/kg. The Hb binding capacity of Apohb‐Hp was 15 mg/mL and the heme binding capacity was 0.5 mg/mL, the Hb binding capacity of Hp was 30 mg/mL with no heme binding capacity. During baseline, hypoxia and normoxia systemic parameters (mean arteriole pressure and heart rate), blood gases (pO2,A, pO2,v, sO2,A­, sO2,v), hematological properties (total Hb concentration, hematocrit, and concentration of lactate) and microcirculatory hemodynamics (velocity, diameter, flow and functional capillary density) were measured. After the mice were sacrificed a count of present macrophages, neutrophiles and lymphocytes were quantified in the plasmaThere was no notable changes in blood gas concentrations with any of the treatments. Apohb‐hp treatment treated animals showed an improvement in microcirculatory dynamics. Apohb‐Hp treated animals showed a statically higher diameter in arterioles (&lt;40 um and &gt;40 um) as well as venules with a diameter between 20‐60 um when compared to controls and hp treated mice, suggesting that treatment prevented vasoconstriction in arterioles. Apohb‐hp treatment also improved flow rates in arterioles and venules when compared to the control and hp treated mice. Furthermore, the functional capillary density of the mice followed for 72 hours after hypoxia showed that the mice treated with apohb‐hp had a greater number of functional capillaries compared to the control and hp treated group. Lastly, the amount macrophages, neutrophiles and lymphocytes decreased when treated with apohb‐hp illustrating another promising result as VOCs are generally associated with an active immune response.These results suggest a role for ApoHb‐Hp complex in alleviating microvascular changes occurring during hypoxia mediated acute vaso‐occlusion in SCD and merits further study for development as a potential therapeutic.

Relationships Between Transcranial Doppler Velocity, Von Willebrand Factor, Factor VIII, and Hematological Parameters in Children with Sickle Cell Anemia: A Comparative Cross-Sectional Study

Background and Objectives Sickle cell anemia (SCA) is associated with recurrent acute inflammatory processes. These inflammatory processes could lead to elevation of Factor VIII and Von Willebrand Factor levels, thereby increasing the risk of stroke in SCA children. This study aims to determine vWF/FVIII levels in children with SCA and their association with abnormal transcranial Doppler (TCD). Subjects and Methods This study enrolled 75 children, including 24 SCA cases with normal TCD, 27 SCA cases with abnormal TCD, and 24 Hb AA controls, all aged between 2 and 16 years. Transcranial Doppler (TCD) ultrasound was performed to measure the cerebral blood velocity. Venous blood drawn from each participant was used to determine the levels of von Willebrand Factor Antigen (vWF: Ag) and Factor VIII (FVIII) and the complete blood count (CBC). Relationships among the measured parameters were determined using SPSS version 25. Statistical significance was set at P &lt; .05. Results FVIII and vWF levels were significantly higher among children with SCA compared to the Hb AA controls ( P &lt; .001). Although SCA patients with abnormal TCD tended to have higher levels of FVIII and vWF, this result did not attain statistical significance ( P &gt; .05). There was a moderate negative correlation between the left middle cerebral artery and FVIII, (r = −0.332; P = .017). Children with SCA showing an abnormal TCD velocity had significantly higher platelet count compared to those with normal TCD ( P = .018). Conclusion Children with SCA have elevated levels of FVIII and vWF, and an abnormal TCD velocity is associated with elevated platelet count.

Determination of Coagulation Profile among Children with Sickle Cell: Anemia in Steady-State and Crisis

Background: Patients with Sickle Cell Disease (SCD) have been found to have an aberrant coagulation profile. One of the primary elements hypothesized to contribute to the vaso-occlusive crisis that characterizes sickle cell disease is coagulopathy (SCD)&#x0D; Material and Methods: A total of 150 children were enrolled as follows, 50 children with Sickle Cell Anemia (SCA) in steady-state, 50 in crisis, and 50 with Hb AA genotype as control. 5 ml of Venous blood was collected. The platelets count was performed using Sysmex KX21N, the electrical impedance principle. The Mean Platelet Volume (MPV) is derived from the impedance platelet size distribution curve. STAGO PT31039352) semi-automated machine was used for estimation of PT, and APTT.&#x0D; Result: children with SCA have significantly a prolonged PT, and APTT compared with children with normal hemoglobin genotype (P. value &lt; 0.001) the mean of PT was (16.64, and 12.6) respectively, and APTT (41.45 and 37.94) consequently. A significant increase in platelet count between patients with SCA when compared with control (p. value 0.02), however a significant prolonged in APTT when compared to steady-state (P. value 0.005). MPV among children with crises when compared with steady-state revealed a significant result (p. value 0.006) the mean of MPV in steady-state = 6.79 while the mean of MPV in crisis = 7.09.&#x0D; Conclusion: children with sickle cell anemia had a longer coagulation profile and marked variation in platelet count, which may increase the risk of thrombosis or bleeding.

Prevalence of Plasmodium Falciparum among Medical Students with different ABO Groups and Electrophoretic Patterns in a Tertiary Institution in Nnewi, Nigeria

There is evidence that Plasmodium falciparum (Pf) malaria is influenced by ABO blood type but the extent of association is not fully established. Some investigators opinioned that haemoglobin electrophoretic patterns are a factor in susceptibility to Pf infection but there is no consensus on possible association between it and ABO blood group and Hb genotypes. This study was designed to determine the prevalence of Pf among different ABO blood groups and Hb electrophoretic patterns of medical students of a tertiary institution in Nnewi, Nigeria. A total of 80 subjects (41 males and 39 females) aged 18-30 years who reported to the Medical Centre of the institution on account of febrile illness were recruited for the study. Information on age, previous malaria episodes and recent use of prophylaxis were sought. Three milliliters (3ml) of blood were collected into EDTA container for ABO grouping, Hb electrophoresis and blood films for P. falciparum detection and quantification by microscopy. Pf prevalence among the subjects was 47.5% (38/80). Thirty-one (38.75%) of the subjects were of blood group O, 27 (33.75%) group A, 19 (23.75%) blood group B and 3(3.75%) blood group AB. Fifty-two (65%) of the subjects were Hb AA and 28 (35%) AS. No significance difference was seen between malaria episodes and ABO blood groups; Hb electrophoretic patterns; gender and parasite density (p&gt;0.05) respectively. A negative correlation was observed between parasite density and age (r= -0.180, p = 0.109). Pf infection, frequency of infection and parasite load is not influenced by blood group and Hb electrophoretic patterns in our study population.

Improved Hemolytic Anemia with GBT1118 Is Reno-Protective in Transgenic Sickle Mice

Intravascular hemolysis releases cell-free hemoglobin and heme into the circulation. When haptoglobin and hemopexin are depleted, as observed in sickle cell anemia, cell-free hemoglobin and heme can cause direct oxidative injury, upregulation of inflammatory and immune response pathways, and depletion of nitric oxide. The kidneys are the primary route for clearance of non-scavenged cell-free hemoglobin and heme and are particularly susceptible to these deleterious pathways (PMID 31455889).In patients with sickle cell anemia, increased markers of hemolysis and hemoglobinuria are risk factors for kidney disease and kidney disease progression (PMID 24329963). Voxelotor is a small molecule allosteric modulator that binds and maintains sickle hemoglobin in the oxygenated state, thereby preventing hemoglobin S polymerization and red blood cell sickling. In a phase 3 study, voxelotor improved the degree of hemolysis, as reflected by a rise in hemoglobin concentration and a reduction in indirect bilirubin and reticulocyte percentage (PMID 31199090). The benefits of reducing hemolytic anemia with voxelotor on kidney function are unknown.We investigated whether GBT1118, an analogue of voxelotor with improved pharmacokinetic properties in transgenic sickle mice, would improve biomarkers of kidney damage and kidney function in sickle cell anemia. Transgenic sickle mice (Hb SS; Townes model, Jackson Laboratory) were treated with either GBT1118 (5 male, 5 female) or control (5 male, 5 female) chow from 12 to 24 weeks of age. Urine was collected using metabolic cages for 24 hours at 3 week intervals and blood was collected at baseline, 6 weeks, and 12 weeks of therapy. We compared markers of hemolysis (hemoglobin, reticulocyte percentage), reactive oxygen species (thibarbituric acid reactive substances [TBARS], kidney injury (kidney injury molecule-1 [KIM-1], nephrin), and kidney function (urine albumin and protein, serum blood urea nitrogen [BUN] and creatinine) at the respective time points using ANOVA and adjusting for gender. Mean and standard error values are provided.Consistent with the clinical results observed in the voxelotor phase 3 study, we observed significant improvements in the hemoglobin concentrations and reticulocyte percentages of Hb SS mice treated with GBT1118 versus control (Figure 1A and 1B). The hemoglobin occupancy of GBT1118 after 6 weeks (33 ± 2%) and 12 weeks (28 ± 2%) of therapy were also similar to what was observed in the phase 3 clinical study. An improvement in hemolysis led to reduced hemoglobinuria (6 weeks: treated = 0.07 ± 0.03 vs. control = 0.72 ± 0.2 ng/day, P = 0.004; 12 weeks: treated = 0.04 ± 0.03 vs. control = 1.06 ± 0.32 ng/day, P = 0.006) and less oxidant damage in the kidneys, assessed by urine TBARS concentration, in the treated versus control mice (6 weeks: treated = 31 ± 6 vs. control = 60 ± 10 nmol/day, P = 0.001; 12 weeks: treated = 29 ± 4 vs. control = 71 ± 9 nmol/day, P < 0.001).At 24 weeks of age, markers of glomerular (nephrin: treated = 3.5 ± 0.7 vs. control = 8.5 ± 1.6 ng/day) and tubular injury (KIM-1: treated = 169.6 ± 19.4 vs. control = 348.9 ± 48.2 pg/day) were significantly improved in the treated versus control Hb SS mice (P< 0.001) (Figure 1C). Albuminuria remained stable in the treated Hb SS mice and was relatively close in value to the degree of albuminuria observed in Hb AA mice (Figure 1D). In contrast, albuminuria progressively increased with older age in the control Hb SS mice. A similar pattern was observed for proteinuria (24 week: Hb SS treated = 2.02 ± 0.17 mg/day, Hb SS control = 3.65 ± 0.44 mg/day, Hb AA = 1.65 ± 0.27 mg/day). At 24 weeks of age, the treated Hb SS mice had serum BUN and creatinine values that were similar to what was observed at 12 weeks of age in the Hb SS mice and to the 24-week old Hb AA mice (Figure 1E, 1F). Furthermore, the values for serum BUN and creatinine were significantly lower in the treated versus control Hb SS mice at 24 weeks of age (P ≤ 0.04).In conclusion, we demonstrate that an improvement in hemolysis results in preserved kidney function in transgenic sickle mice. Our findings highlight the clinical importance of hemolytic anemia in the pathophysiology of sickle cell nephropathy. Our results also provide support for developing strategies to mitigate hemolysis in sickle cell anemia in order to provide a targeted approach to improve kidney disease, a devastating complication associated with high morbidity and mortality, in sickle cell anemia. [Display omitted] DisclosuresGordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding.

Haemoglobin Type, Haematology and Morphometric Characteristics of Muscovy Ducks Reared in Calabar, Nigeria

This study determined the haemoglobin polymorphism, haematology and morphometric characteristics of Muscovy ducks. A total of 80 adult Muscovy ducks (40 per sex) respectively were used for the study which lasted 60 days. Blood samples of the ducks were collected and analysed for haemoglobin type and haematological traits while body weight (BWT) and body parts such as body girth (BG), wing length (WL), keel length (KL), shank length (SL), body length (BL), thigh length (TL) and Body circumference (BC) were taken and subjected to one –way ANOVA. Drakes exhibited significantly (P&lt;0.05) higher values in body girth, body length and thigh length than the ducks. Average values recorded were BWT (2040 g), BC (30.43cm), KL (25.75cm), BL (25.78cm) and TL (4.85cm). Haemoglobin types found among the ducks were Hb AA, AB and BB, males being predominantly of Hb BB and females of Hb AA. Gene frequencies of 0.5435 and 0.4565 were recorded for alleles A and B respectively among the ducks. Genotype frequencies were AA (39.14%), AB (30.43%) and BB (30.43%). Some average haematological values recorded were Packed Cell Volume (PCV) (29.86%), Erythrocytes Sedimentation Rate (ESR) (7.34mm/hr), Haemoglobin Concentration (HBC) (11.93g/dl), Red Blood Cell Counts (RBC) (2.4 × 106/L) and White Blood Cell (WBC) (1.98 × 103/L). Results of body morphometry from this research could serve as an important basis for characterization, conservation and improvement of this duck breed in the study location. The establishment of the haemoglobin types of ducks in Calabar is a precursor to the determination of its relationship(s) with economic traits in the birds. Haematological values recorded in this study showed that the ducks were in normal condition of health and growth.

Topographical Differences in Orexin Activation after Pain Assessments in Hb AA and HbSS Sickle Mice

The orexins are neuropeptides that are synthesized in the perifornical area (PFA), lateral hypothalamus (LH) and dorsomedial hypothalamus (DMH) and mediate arousal, sleep, energy balance, food and drug reward. The LH region is associated with reward, while the PFA and DMH are more associated with stress and arousal. Data suggest that orexin-A has an analgesic effect on inflammatory pain and it may affect mechanisms underlying the maintenance of neuropathic pain. To date, there have been no published studies to investigate this system as a potential target to treat neuropathic pain in a mouse model of sickle cell disease (SCD). Therefore, we seek to determine activational and topographical differences in neuronal activation of the orexin system in a mouse model of SCD by using an array of behavioral measures to characterize pain in transgenic mice expressing human hemoglobin.Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS) and age-/gender-matched controls (HbAA mice; n = 5/group, 20-30g) expressing normal human hemoglobin A are used in the study. All behavioral tests are performed in a quiet room maintained at a constant temperature of 23- 25°C. All mice are habituated to each test protocol and environment for at least three consecutive days before collecting baseline measurements and testing. Three measures are used to assess pain-related behaviors: thermal/heat hyperalgesia (test for heat sensitivity), cold hyperalgesia (for cold sensitivity), and mechanical hyperalgesia (sensitivity to mechanical stimulus). Ninety minutes after the pain assessment testing, brains from HbAA and HbSS mice are quick frozen and hypothalamic brain sections from the mice are processed to visualize orexin-A and c-Fos using double label immunohistochemistry.Preliminary analyses show that significant topographical differences are observed in LH versus PFA for HbAA and HbSS mice (p<0.05) and LH versus DMH for HbAA and HbSS mice (p<0.05). This indicates that the number of double labeled neurons in the LH is significantly lower than the number of double labeled neurons in the PFA and DMH of HbAA and HbSS mice. Additionally, a significant topographical difference is observed in LH versus DMH for only HbSS mice (p<0.05). These data demonstrate that the number of activated orexin neurons is greater after pain testing in the DMH (a region more associated with stress and arousal) versus LH in HbSS mice. There is a trend towards a significant increase in activated LH orexin neurons in HbAA versus HbSS mice. Additional studies are underway to determine whether fewer reward-associated, orexin neurons in the LH are activated in HbSS versus HbAA mice after pain testing, and whether this decrease in orexin activation may provide a possible explanation for the increased hyperalgesia in HbSS sickle mice, which contributes to neuropathic pain observed in SCD. DisclosuresGupta:Tautona Group: Consultancy, Honoraria; Fera Pharmaceuticals LLC: Consultancy, Honoraria.

Hemechip: An Automated Portable Microchip Electrophoresis Platform for Point-of-Care Sickle Cell Disease Screening

Sickle Cell Disease (SCD) affects more than 14 million people globally, primarily in economically disadvantaged populations. Its management requires early diagnosis, monitoring, and treatment throughout the lifespan of the patient. Early diagnosis of SCD remains a challenge in the developing world due to requirements for sophisticated lab equipment and skilled personnel. American Society of Hematology with other organizations launched the Sickle Cell Disease Coalition, calling for improved patient care. In low-income countries, notably those in Sub-Saharan Africa, the mortality rate for children <5 years with SCD is 50-80%, without early diagnosis and basic care. Access to testing is lacking in these areas since existing SCD screening methods are expensive and resource intensive. The situation calls for an accurate and affordable diagnostic tool designed for point-of-care (POC). To address this need, we developed HemeChip, an accurate, affordable, and rapid “microchip electrophoresis” test, packaged in a small, easy-to-use, and low-cost mass-producible platform. HemeChip identifies SCD and other critical hemoglobinopathies as well as provides relative percentages of hemoglobin types. HemeChip is the first paper-based microchip electrophoresis platform that performs both qualitative and quantitative analysis of hemoglobins. Here, we build on our previous work (Blood 2015 126:3379) by presenting an initial clinical performance analysis of HemeChip's results, compared with High Performance Liquid Chromatography (HPLC), the clinical gold standard.The compact single-use microchip of the HemeChip platform encompasses cellulose acetate paper, in which the hemoglobin separation takes place, as well as integrated stainless-steel electrodes (Fig. 1A). The HemeChip test is performed in four steps: (i) finger/heel prick equivalent amount of whole blood (diluted and lysed) is applied to the microchip (Fig. 1A); (ii) microchip is placed inside the HemeChip reader (Fig. 1B); (iii) real time images of the hemoglobin electrophoresis are captured during the test by the portable reader; and (iv) custom built-in software automatically analyses the results real-time, extracts relevant peak positions, and performs quantitative analysis (Fig. 1C) similar to HPLC. Total procedure takes less than 10 minutes.HemeChip reliably detects hemoglobin types and categorizes patient samples as normal (Hb AA), sickle cell trait (Hb SA), sickle cell anemia (Hb SS), and sickle-hemoglobin C disease (Hb SC) (Table 1). All 77 blood samples tested were obtained under Institutional Review Board (IRB) approval. Tests were performed by both experienced and minimally-trained personnel. Test results for both categories did not introduce discrepancy or variation in accuracy. The HemeChip tests correctly categorized patient samples as Hb AA, Hb SA, Hb SS, and Hb SC with 96.6%-100% accuracy compared with HPLC (Table 1).HemeChip provides high accuracy for hemoglobin phenotype detection and excellent compliance with the clinical gold standard for quantification of the results, which suggests that the HemeChip platform could address the unmet need for screening in regions where SCD is prevalent. Field-testing of HemeChip for SCD screening is ongoing in sub-Saharan Africa.Acknowledgments: This work was supported by: Case-Coulter Translational Research Partnership, Clinical and Translational Science Collaborative of Cleveland (National Center for Advancing Translational Sciences), NIH Center for Accelerated Innovation at Cleveland Clinic, and Center for Integration of Medicine & Innovative Technology.Figure 1: HemeChip overview and initial clinical testing results. (A) Illustration of hemoglobin separation during a HemeChip test. (i) initial, (ii) intermediate, and (iii) final phase of the separation in HemeChip. (B) (i) Portable HemeChip Reader with custom built-in analysis software, (ii) Ssingle use HemeChip cartridge microchip after a test. (C) HemeChip image analysis showing identification and quantification for an Hb SA blood sample. (i) Captured image of hemoglobin separation. (ii) Averaged pixel plot along the HemeChip length reveals intensity peaks and hemoglobin types. (iii) Baseline corrected area under curve determines the relative percentage of hemoglobins compared with HPLC. [Display omitted] DisclosuresLittle:Hemex Health: Equity Ownership. Gurkan:Hemex Health: Employment, Equity Ownership.