Quantitative Model-Based Assessment of Multiple Sickle Cell Disease Therapeutic Approaches Alone and in Combination.

Three sickle cell disease (SCD) treatment strategies, stabilizing oxygenated hemoglobin (oxyHb), lowering 2,3-BPG, and inducing fetal hemoglobin (HbF) expression aim to prevent red blood cell (RBC) sickling by reducing tense-state sickle hemoglobin that contributes to polymer formation. Induction of 30% HbF is seen as the gold standard because 30% endogenous expression is associated with a lack of symptoms. However, the level of intervention required to achieve equivalent polymerization protection by the other strategies is uncertain, and there is little understanding of how these approaches could work in combination. We sought to develop an oxygen saturation model that could assess polymerization protection of all three approaches alone or in combination by extending the Monod-Wymann-Changeux model to include additional mechanisms. Applying the model to monotherapies suggests 51% sickle hemoglobin (HbS) occupancy with an oxyHb stabilizer or lowering RBC 2,3 BPG concentrations to 1.8 mM would produce comparable polymerization protection as 30% HbF. The model predictions are consistent with observed clinical response to the oxyHb stabilizer voxelotor and the 2,3-BPG reducer etavopivat. The model also suggests combination therapy will have added benefit in the case of dose limitations, as is the case for voxelotor, which the model predicts could be combined with 20% HbF or 2,3-BPG reduction to 3.75 mM to reach equivalent protection as 30% HbF. The proposed model represents a unified framework that is useful in supporting decisions in preclinical and early clinical development and capable of evolving with clinical experience to gain new and increasingly confident insights into treatment strategies for SCD.

A low-cost miniature immunosensor for haemoglobin as a device for the future detection of gastrointestinal bleeding.

Gastrointestinal bleeding (GIB) is a serious medical condition, which requires immediate attention to establish the cause of the bleeding. Here, we present the development of a miniaturised electrochemical impedance spectroscopy (EIS) device for the detection of GIB. The device performs EIS measurements up to 100 kHz. Following the development of an immunosensor for haemoglobin (Hb) on screen printed electrodes, the EIS device was used for detecting Hb as an early indication of bleeding. The sensor was able to detect Hb in a redox solution in a linear range between 5 µg mL-1 and 60 µg mL-1, with a limit of detection of 13.3 µg mL-1. It was also possible to detect Hb in simulated intestinal fluid, without the need for a redox solution, within a range of 10 µg mL-1 to 10 mg mL-1 with a limit of detection of 2.31 mg mL-1. The miniature EIS device developed in this work is inexpensive, with an estimated cost per unit of £30, and has shown a comparable performance to existing commercial tools, demonstrating its potential to be used in the future as an ingestible sensor to detect GIB. All these measurements were carried out in a purpose built flow cell with supporting hardware electronics outside the cell. Integration of the hardware and the sensing electrodes was demonstrated in pill form. This pill after integration sampling fluidics has potential to be used in detecting gastrointestinal bleeding.

Recommendations for proficiency testing criteria for hemoglobin A1c based on the Shanghai Center for Clinical Laboratory's study.

OBJECTIVES: The US Centers for Medicare & Medicaid Services proposed in 2019 that glycated hemoglobin A1c (HbA1c) be a CLIA'88 regulated analyte. People who commented expressed concerns that the proposed acceptance limit (AL, HbA1c in NGSP unit) ±10% for proficiency testing (PT) would be unable to maintain already improved analytical performance and guarantee the clinical utility of HbA1c testing. Assessing impact of various ALs on PT performance is needed to provide scientific evidence for adopting an appropriate AL. METHODS: Ten patient EDTA-whole blood specimens were distributed to 318 and 336 laboratories in the 2018 and 2019 PT events organized by Shanghai Center for Clinical Laboratory (SCCL). HbA1c concentrations were measured by participants using various methodologies commonly used in the USA and China. Targets were determined using secondary reference measurement procedures (SRM) at SCCL. "Failed Results" were those outside the SRM-defined target ± AL (5% through 10%). Laboratories with Failed Results ≥2 out of five samples per PT event obtained Event Unsatisfactory Status. RESULTS: HbA1c target values ranged 33.3 mmol/mol (5.2 NGSP%) -102.2 mmol/mol (11.5 NGSP%) for 2018 event, and 33.3 mmol/mol (5.2 NGSP%) -84.7 mmol/mol (9.9 NGSP%) for 2019 event. Overall Laboratory Event Unsatisfactory Rates were 11.3-12.2%, 4.8-5.3%, 0.9-3.1%, 0.6-2.2%, 0.6-1.4% and 0.6-1.4%, at AL of ±5, ±6, ±7, ±8, ±9 and ±10%, respectively. CONCLUSIONS: The AL (in NGSP unit) of ±6% or ±7% for PT evaluation of HbA1c results would be appropriate, with satisfactory event scores for about 95% of participant laboratories in a PT event.

Increased Hemoglobin A1c Time in Range Reduces Adverse Health Outcomes in Older Adults With Diabetes.

OBJECTIVE: Short- and long-term glycemic variability are risk factors for diabetes complications. However, there are no validated A1C target ranges or measures of A1C stability in older adults. We evaluated the association of a patient-specific A1C variability measure, A1C time in range (A1C TIR), on major adverse outcomes. RESEARCH DESIGN AND METHODS: We conducted a retrospective observational study using administrative data from the Department of Veterans Affairs and Medicare from 2004 to 2016. Patients were ≥65 years old, had diabetes, and had at least four A1C tests during a 3-year baseline period. A1C TIR was the percentage of days during the baseline in which A1C was in an individualized target range (6.0-7.0% up to 8.0-9.0%) on the basis of clinical characteristics and predicted life expectancy. Increasing A1C TIR was divided into categories of 20% increments and linked to mortality and cardiovascular disease (CVD) (i.e., myocardial infarction, stroke). RESULTS: The study included 402,043 veterans (mean [SD] age 76.9 [5.7] years, 98.8% male). During an average of 5.5 years of follow-up, A1C TIR had a graded relationship with mortality and CVD. Cox proportional hazards models showed that lower A1C TIR was associated with increased mortality (A1C TIR 0 to <20%: hazard ratio [HR] 1.22 [95% CI 1.20-1.25]) and CVD (A1C TIR 0 to <20%: HR 1.14 [95% CI 1.11-1.19]) compared with A1C TIR 80-100%. Competing risk models and shorter follow-up (e.g., 24 months) showed similar results. CONCLUSIONS: In older adults with diabetes, maintaining A1C levels within individualized target ranges is associated with lower risk of mortality and CVD.

Análise do perfil de doadores de sangue positivos para hemoglobina S em hemocentro do estado do Rio Grande do Sul / Profile assessment of blood donors with sickle cell trait in a public blood bank in Southern Brazil

Introdução: Portadores do traço falciforme podem doar sangue, porém requerem maior atenção ao direcionamento da sua transfusão. Considerando o perfil étnico- racial da região sul do Brasil, o presente artigo teve como objetivo analisar o perfil e a prevalência de Hemoglobina S em um hemocentro público de Porto Alegre. Métodos: Estudo transversal retrospectivo realizado através de uma pesquisa em banco de dados cadastrais e de resultados de testes imunológicos no período de janeiro de 2015 a dezembro de 2019. Resultados: Foram obtidos um total de 8.2363 registros cadastrais e 6.7184 testes imunológicos. Dos testes, 467 foram positivos para Hemoglobina S de 134 doadores distintos. O percentual de Hb S positiva apresentou uma média de 0,7% anual entre todos os doadores. Entre doadores autodeclarados "Negros" a prevalência é de 0,92% e "Caucasianos" é de 0,13%. Conclusão: Os dados corroboram com a literatura, porém o espectro social que abrange as denominações "Caucasiano Brasileiro" e "Mestiço" permanecem em questionamento dentro da relevância do marcador étnico da Hemoglobina S no Rio Grande do Sul. (AU)

Introduction: People with sickle cell trait can donate blood, but special attention should be paid to the transfusion recipient. Considering the ethnic-racial profile of Southern Brazil, this article aimed to analyze the profile and prevalence of hemoglobin S in a public blood bank in Porto Alegre. Methods: A quantitative, retrospective, and cross-sectional study was conducted to assess the profile of blood donors positively screened for hemoglobin S from January 2015 to December 2019 in a public blood bank in Southern Brazil. Results: A total of 82,363 records and 67,184 immunohematological tests were obtained. Regarding the tests, 467 were positive for hemoglobin S among 134 different donors. The percentage of positive hemoglobin S has remained stable over the years, with an annual average of 0.7%. The prevalence of self-reported "black" and "Brazilian Caucasian" blood donors was 0.92% and 0.13%, respectively. Conclusions: The data are in accordance with the literature; however, the social spectrum that comprises the terms "Brazilian Caucasian" and "mixed-race" remains in question regarding the relevance of the ethnic marker of hemoglobin S in Southern Brazil. (AU)

In vitro assessment of the anti-sickling properties of Buchholzia coriacea and Mucuna pruriens seed extracts.

Sickle cell disease is a group of diseases inherited through the gene and it affects the haemoglobin in the red blood cell. This study investigated the methanol seed extract of Buchholzia coriacea for possible in vitro anti-sickling effects and also determined the effect of Mucuna pruriens seed extract on the solubility and oxygen-binding rate of sickle cell haemoglobin. Sickle cell blood was collected from sickle cell disease patients with subsequent addition of 2% sodium metabisulphite to cause more sickling. Varying concentrations of the seed extracts (50%, 25%, 12.5% and 6.25%) were added to the pre-treated blood for these in vitro assays. The results showed that the extract of Buchholzia coriacea significantly (P < 0.05) inhibited sickling at all concentrations with the highest percentage inhibition of 73.3 ± 5.8, reversed sickled erythrocytes at all concentrations with the highest percentage reversal of 83.3 ± 5.8 and significantly (P < 0.05) inhibited polymerisation at all concentrations used in comparison to the parallel control. The extract of Mucuna pruriens seed significantly (P < 0.05) increased the solubility of sickle haemoglobin at 50%, 25%, 12.5% and 6.25% concentrations, increased Fe2+/Fe3+ ratio from 1.7 (control) to 12.2 (50% concentration) and reduced osmotic fragility (at 12.5% and 6.25% concentrations) when compared with parallel control. The results indicate the feasibility of the seed extracts as promising agents in the management of sickle cell disease.

Confined filaments in soft vesicles - the case of sickle red blood cells.

Abnormal shapes of red blood cells (RBC) have been associated with various diseases. Diverse RBC shapes have also been intriguing for membrane biophysics. Here we focus on sickle shaped RBC which form due to abnormal growth of semi-rigid hemoglobin (HbS) fibers confined in RBC. Using the area difference elasticity (ADE) model for RBC and worm-like chain model for the confined HbS fibers, we explore shape deformations at equilibrium using Monte-Carlo simulations. We show that while a single HbS fiber is not rigid enough to produce sickle like deformation, a fiber bundle can do so. We also consider multiple disjoint filaments and find that confinement can generate multipolar RBC shapes and can even promote helical filament conformations which have not been discussed before. We show that the same model, when applied to microtubules confined in phospholipid vesicles, predicts vesicle tubulation. In addition we reproduce the tube collapse transition and tennis racket type vesicle shapes, as reported in experiments. We conclude that with a decrease in the surface area to volume ratio, and membrane rigidity, the vesicles prefer tubulation over sickling. The highlight of this work is several important non-axisymmetric RBC and vesicle shapes, which have never been explored in simulations.

Anemia Screening in Naval Aviation: Is Hemoglobin a Better Indicator Than Hematocrit as the Primary Index?

INTRODUCTION: Because of the rigorous mental and physical health requirements for Naval Aviation, all applicants and designated personnel must meet physical standards, including initial and periodic screening for anemia. Most standards, including for accession to the U.S. Navy, use hemoglobin as the standard marker to screen for anemia. Moreover, previous literature generally supports the assertion that hemoglobin is more reliable and accurate than hematocrit. However, the U.S. Navy Aeromedical Reference and Waiver Guide uses a hematocrit standard for anemia screening. The purpose of this study was to determine whether hemoglobin or hematocrit correlates better with clinical anemia and evaluate which index is a more accurate indicator for anemia screening in Naval Aviation personnel. MATERIALS AND METHODS: This is a retrospective cross-sectional study of Naval Aviation applicants (N = 95) who were evaluated by the Human Performance and Aeromedical Qualifications department at Naval Aerospace Medical Institute Clinic in Pensacola, Florida, from January 1, 2015 to September 30, 2018. Data were collected from electronic medical records in a de-identified manner that included demographics, class designations, labs results, diagnoses, and final disposition. Logistic regression was used to analyze whether hemoglobin (using the U.S. Navy standard of 13.5 g/dL for men and 12.0 g/dL for women) or hematocrit (using the Naval Aviation standard of 40% for men and 37% for women) predicted the diagnosis of anemia for subjects having at least one lab sample (1-sample) and for those having three samples (3-samples). Sensitivity and specificity values were calculated for hemoglobin and hematocrit as tools to predict a diagnosis of anemia using the same standards in the 1-sample and 3-sample groups. RESULTS: Data were collected for 95 subjects, 53 of whom had three sets of paired hemoglobin/hematocrit values. Using logistic regression, hemoglobin was found to be a statistically significant predictor of anemia for both the 1-sample group (odds ratio 3.4, confidence interval [1.130-10.196], P < 0.05) and the 3-sample group (odds ratio 10.5, confidence interval [1.776-62.580], P < 0.01). Hematocrit was not a significant predictor in either group. Hemoglobin was 80% sensitive and 52.3% specific for a diagnosis of anemia in the 1-sample group and 91.3% sensitive and 50.0% specific in the 3-samples group. Hematocrit was 86.7% sensitive and 35.4% specific for a diagnosis of anemia in the 1-sample group and 91.3% sensitive and 23.3% specific in the 3-samples group. CONCLUSIONS: This study found that hemoglobin correlates better with the diagnosis of anemia than hematocrit. When three samples are analyzed, hemoglobin is equally sensitive and more specific than hematocrit. Based on these results and the U.S. Navy accession standards using hemoglobin as the standard index for anemia, the U.S. Navy Aeromedical Reference and Waiver Guide should consider using hemoglobin instead of hematocrit to screen for anemia. Future research should focus on prospective research to determine whether hemoglobin or hematocrit is a better indicator of anemia in screening military personnel.

High-throughput assessment of hemoglobin polymer in single red blood cells from sickle cell patients under controlled oxygen tension.

Sickle cell disease (SCD) is caused by a variant hemoglobin molecule that polymerizes inside red blood cells (RBCs) in reduced oxygen tension. Treatment development has been slow for this typically severe disease, but there is current optimism for curative gene transfer strategies to induce expression of fetal hemoglobin or other nonsickling hemoglobin isoforms. All SCD morbidity and mortality arise directly or indirectly from polymer formation in individual RBCs. Identifying patients at highest risk of complications and treatment candidates with the greatest curative potential therefore requires determining the amount of polymer in individual RBCs under controlled oxygen. Here, we report a semiquantitative measurement of hemoglobin polymer in single RBCs as a function of oxygen. The method takes advantage of the reduced oxygen affinity of hemoglobin polymer to infer polymer content for thousands of RBCs from their overall oxygen saturation. The method enables approaches for SCD treatment development and precision medicine.

Genetic Background of ß-Thalassemia in Northeast Algeria with Assessment of the Thalassemia Severity Score and Description of a new ß0-Thalassemia Frameshift Mutation (HBB: c.374dup; p.Pro126Thrfs*15).

ß-Thalassemia (ß-thal) is a genetic disorder representing a major health problem in Algeria. Our first objective was to determine the allelic frequencies and molecular spectrum of ß-thal mutations in patients with major hemoglobinopathies [ß-thal major (ß-TM) and sickle cell disease] in three provinces of northeast Algeria. Our second objective was to assess if the clinical management of ß-TM patients depended on their region of origin. Our last objective was to assess a population originating from Maghreb, the reliability of the thalassemia severity score (TSS) for patients with homozygous ß-thal. Sanger HBB gene sequencing was performed on 59 patients with sickle cell disease and 60 with ß-TM. For the latter patients, the genetic modifiers of the TSS were genotyped: α-thalassemia (α-thal) deletions and four Hb F-inducing polymorphisms (XmnI, rs1427407 and rs10189857 for BCL11A and rs9399137 for HMIP). Eleven different ß-thal mutations were found but two of them (HBB: c.118C>T and HBB: c.93-21G>A) accounted for about 70.0% of the ß-thal alleles. A relatively high proportion of Hb S (HBB: c.20A>T)/ß-thal genotypes (27.0%) was found in our sickle cell disease cohort where a new frameshift ß0-thal mutation (HBB: c.374dup; p.Pro126Thrfs*15) was identified. No difference was found in the three provinces. Of the 60 ß-TM patients, those with a high or very high TSS were significantly younger at the age of first transfusion, thus assessing the reliability of this scoring system in a Maghrebin cohort. Trends for a lower age of splenectomy and high ferritin levels were also detected for the higher TSS categories.

Assessment of iron status and interplay between lipid peroxidation and antioxidant capacity in common hemoglobin variants in Osun State, southwestern Nigeria.

Hemoglobin (Hb) and iron are prooxidants in nature and sources of free radicals in the biological system of all Hb phenotypes. Recent evidence linked abnormal hemoglobin S and C (HbSC) in sickle cell disease (SCD) to various complications in multiple oxidative processes. However, similar studies in relation to abnormal Hb traits are sparse. Besides, reports on activities of antioxidant enzymes and iron status in SCDs are still contradictory. This study assessed the interplay between lipid peroxidation and antioxidant defense capacity in various Hb variants. We enrolled 193 participants with different Hb phenotypes. They were consecutive patients with sickle cell anemia (HbSS, n = 32) and hemoglobin SC (HbSC) disease (n = 28) regularly followed up in a steady state. Other participants were subjects with abnormal Hb traits (HbAS, n = 50; HbAC, n = 33) and normal controls (HbAA, n = 50). The hematocrit (Hct) level, hemoglobin (Hb) concentration, iron status, and biochemical parameters including malondialdehyde (MDA), total antioxidant status (TAS), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes were investigated simultaneously. The MDA and SOD levels were significantly higher (P < 0.05) in Hb variants in order of HbSS>HbSC>HbAC>HbAS when compared with controls. Conversely, GPx and TAS levels showed significant reductions (P < 0.05). Similarly, Hct, Hb, and iron concentrations showed significant reductions (P < 0.05) sequentially following HbAC > HbAS > HbSC > HbSS compared with controls. The results suggest that both SCDs and the carriers were relatively more vulnerable to systemic oxidative stress against normal phenotype, and may be owing to ineffective antioxidant mechanisms needed for keeping spontaneous generations of free radicals in control without necessarily iron-mediated.

Point-of-care screening for sickle cell disease in low-resource settings: A multi-center evaluation of HemoTypeSC, a novel rapid test.

Sickle cell disease (SCD) is a common, life-threatening genetic disorder that is best managed when diagnosed early by newborn screening. However, SCD is most prevalent in low-resource regions of the world where newborn screening is rare and diagnosis at the point-of-care is challenging. In many such regions, the majority of affected children die, undiagnosed, before the age of 5 years. A rapid and affordable point-of-care test for SCD is needed. The diagnostic accuracy of HemoTypeSC, a point-of-care immunoassay, for SCD was evaluated in individuals who had SCD, hemoglobin C disease, the related carrier (trait) states, or a normal hemoglobin phenotype. Children and adults participated in low-, medium- and high-resource environments (Ghana [n = 383], Martinique [n = 46], and USA [n = 158]). Paired blood specimens were obtained for HemoTypeSC and a reference diagnostic assay. HemoTypeSC testing was performed at the site of blood collection, and the reference test was performed in a laboratory at each site. In 587 participants, across all study sites, HemoTypeSC had an overall sensitivity of 99.5% and specificity of 99.9% across all hemoglobin phenotypes. The test had 100% sensitivity and specificity for sickle cell anemia. Sensitivity and specificity for detection of normal and trait states were >99%. HemoTypeSC is an inexpensive (<$2 per test), accurate, and rapid point-of-care test that can be used in resource-limited regions with a high prevalence of SCD to provide timely diagnosis and support newborn screening programs.

[Targeted newborn screening for sickle-cell anemia: Sickling test (Emmel test) boundaries in the prenatal assessment in West African area]. / Dépistage néonatal ciblé de la drépanocytose : limites du test de falciformation (test d'Emmel) dans le bilan prénatal en zone ouest africaine.

BACKGROUND: Newborn screening for sickle cell anemia is necessary in Africa where the disease is more frequent. Hemoglobin electrophoresis is used for screening, but is limited by a high cost and difficult access. Sickling test (Emmel test), which is more affordable and technically more accessible, is often requested for prenatal assessment of pregnant women in West African areas to reserve screening for newborns from mothers in whom the positive sickling test attests the presence of hemoglobin S. This study aims to evaluate the number of undetected sickle cell anemia newborns by a screening policy targeting only newborns from mothers in whom a sickling test would have been positive. METHODS: From 2010 to 2012, in Bamako, Mali, West Africa, 2489 newborns were routinely screened for sickle cell anemia at the umbilical cord or heel by isoelectrofocusing and, if necessary, by high-performance liquid chromatography. These newborns were born from 2420 mothers whose hemoglobin was studied by isoelectrofocusing. The data was recorded and processed using Excel software version 14.0.0. We calculated the frequency of the sickle cell gene in mothers and newborns as well as the number of SCA newborns from heterozygous or C homozygous mothers. RESULTS: Of the 2489 newborns, 16 had sickle cell anemia (6 SS and 10 SC); 198 had the sickle cell trait; 139 were AC and 1 was CC. Of the 10 newborns with SC profile, 3 were born from mothers not carrying the S gene but the C gene of hemoglobin and in which an Emmel test would have been negative. CONCLUSION: Targeted newborn screening, based on the results of sickling test in pregnant women, would misdiagnose more than one of six sickle cell anemia newborns who would not benefit from early care. Cost-effectiveness studies of routine newborn screening for sickle cell anemia should lead to a better screening strategy in contexts where hemoglobin S and other hemoglobin defect genes coexist.

Automated RBC exchange compared to manual exchange transfusion for children with sickle cell disease is cost-effective and reduces iron overload.

BACKGROUND: Chronic transfusion in sickle cell disease (SCD) remains the gold standard therapy for stroke prevention and for patients with severe disease despite adequate hydroxyurea treatment. The aim of our study was to assess the safety and efficacy of automated red blood cell exchange (aRBX) in patients with SCD previously treated with manual exchange transfusion (MET). Costs related to transfusion and chelation overtime were evaluated. STUDY DESIGN AND METHODS: Beginning in January 2012, children with SCD who weighed 30 kg or more on MET could switch to aRBX. Clinical, biological, and procedures' data, including costs, were recorded for the last 6 months on MET and compared to those after the first and the second year on aRBX. RESULTS: Ten patients switched from MET to aRBX at a median age of 11.8 years. After the switch, median hemoglobin S (HbS) increased significantly (33.5% on MET compared to 45% on aRBX; p < 0.001) but remained in the target values for all patients. Median ferritin decreased significantly (663.3 µg/L on MET compared to 126.8 µg/L on aRBX; p < 0.001) and intervals between procedures were significantly longer. The requirements of red blood cells (RBCs)/kg/year were not different on MET (0.88 unit/kg/year) than during the second year on aRBX (1.07 unit/kg/year; p = NS). MET costs were similar compared to aRBX since chelation was stopped in previously treated patients. CONCLUSION: Erythrocytapheresis reduces iron overload and allows a longer interval between procedures without a higher RBC requirement from the second year on aRBX. The cost did not increase as estimated in our Belgian Health Care System.

Single tube allele specific PCR: a low cost technique for molecular screening of sickle cell anaemia in Nigeria.

BACKGROUND: Sickle cell anaemia (SCA) is a hereditary blood disorder caused by a single mutation in the haemoglobin gene. The disease burden of SCA is highest in Nigeria. The allele specific polymerase chain reaction (ASPCR) method is applicable for the direct detection of known single nucleotide polymorphisms (SNPs). OBJECTIVE: To investigate the use of the single tube ASPCR as an accurate and affordable method for SCA screening in Nigeria. METHODS: DNA was extracted from study subjects with normal haemoglobin, HbAA (20), sickle cell anaemia, HbSS (20) and carriers, HbAS (1). Haemoglobin was genotyped by ASPCR using two primer sets that amplifies the wildtype and mutant haemoglobins in each sample. Amplicon sizes were analyzed by gel electrophoresis. RESULTS: Amplicons were visible after electrophoresis at regions 517 base pair (bp) for HbA and 267 bp for HbS. ASPCR correctly and unambiguously detected the presence or absence of haemoglobins A and S from all samples collected, demonstrating its accuracy and precision for the screening of SCA. CONCLUSION: This study validates ASPCR as an effective, low cost approach for the clinical screening of SCA in Nigeria. ASPCR is also applicable for other genetic diseases, paternity testing, and forensics where more expensive fluorescence-based approaches are not obtainable.

Towards a point-of-care strip test to diagnose sickle cell anemia.

A rapid test to identify patients with sickle cell disease could have important benefits in low-resource settings. Sickle cell anemia (SCA) affects about 300,000 newborns each year, the majority of whom are born in sub-Saharan Africa. Low-cost therapies are available to treat SCA, but most countries in sub-Saharan Africa lack robust neonatal screening programs needed to identify patients in need of treatment. To address this need, we developed and evaluated a competitive lateral flow assay that identifies patients with SCA (genotype HbSS) in 15 minutes using undiluted whole blood. A small volume of blood (0.5 µL- 3 µL) is mixed with antibody-coated blue latex beads in a tube and applied to the strip. Strips are then placed in a well of running buffer and allowed to run for 10 minutes. Laboratory evaluation with samples containing different proportions of hemoglobin A (HbA) and hemoglobin S (HbS) indicated that the test should enable identification of SCA patients but not persons with sickle cell trait (SCT). We evaluated the test using 41 samples from individuals with SCA, SCT, and normal blood. With visual inspection or quantitative analysis, we found a 98% accuracy when differentiating SCA from normal and SCT samples as a group (90% sensitivity and 100% specificity for identifying SCA). This work demonstrates important steps towards making a lateral flow test for hemoglobinopathies more appropriate for point-of-care use; further work is needed before the test is appropriate for clinical use.

The Assessment and Sustainable Management of Sickle Cell Disease in the Indigenous Tharu Population of Nepal.

Sickle cell disease is an inherited hemoglobinopathy associated with significant morbidity and mortality. Reports suggest a high sickle cell disease burden among the indigenous Tharu population of Nepal, who for centuries have inhabited regions where malaria is endemic. Unfortunately, health care resources are limited and often inaccessible for Tharu individuals suffering from sickle cell disease. We conducted a large-scale screening effort to estimate the prevalence of Hb S (HBB: c.20A>T) among the Tharu population and delivered community-based education sessions to increase sickle cell disease awareness. A total of 2899 Tharu individuals aged 6 months to 40 years in the rural district of Dang in Western Nepal were screened using a sickling test, of whom, 271 [9.3%; 95% confidence interval (95% CI): 8.3-10.4%] screened positive for Hb S. Those who screened positive were offered diagnostic gel electrophoresis testing. Of the 133 individuals who underwent diagnostic testing, 75.9% (n = 101) were confirmed to be Hb AS heterozygotes, 4.5% (n = 6) were confirmed to be Hb SS homozygotes and 19.5% (n = 26) were false positives. These findings support a large burden of sickle cell disease among the Tharu population and highlight the importance of appropriate resource allocation and management. With a positive predictive value of 80.0% (95% CI: 73.0-87.0%), the sickling test in conjunction with raising local sickle cell disease awareness may be a simple and sustainable way to promote access to health resources.

Interim assessment of liver damage in patients with sickle cell disease using new non-invasive techniques.

We explored transient elastography (TE) and enhanced liver fibrosis (ELF™ ) score with standard markers of liver function to assess liver damage in 193 well patients with sickle cell disease (SCD). Patients with HbSS or HbSß0 thalassaemia (sickle cell anaemia, SCA; N = 134), had significantly higher TE results and ELF scores than those with HbSC (N = 49) disease (TE, 6·8 vs. 5·3, P < 0·0001 and ELF, 9·2 vs. 8·6 P < 0·0001). In SCA patients, TE and ELF correlated significantly with age and all serum liver function tests (LFTs). Additionally, (weak) positive correlation was found with lactate dehydrogenase (TE: r = 0·24, P = 0·004; ELF: r = 0·26 P = 0·002), and (weak) negative correlation with haemoglobin (TE: r = -0·25, P = 0·002; ELF: r = -0·25 P = 0·004). In HbSC patients, correlations were weaker or not significant between TE or ELF, and serum LFTs. All markers of iron loading correlated with TE values when corrected for sickle genotype (serum ferritin, ß = 0·25, P < 0·0001, total blood transfusion units, ß = 0·25, P < 0·0001 and LIC ß = 0·32, P = 0·046). The exploratory study suggests that, while TE could have a role, the utility of ELF score in monitoring liver damage in SCD, needs further longitudinal studies.