Using DNA testing for the precise, definite, and low-cost diagnosis of sickle cell disease and other Haemoglobinopathies: findings from Tanzania

Background Sickle cell disease (SCD) is an important cause of under-five mortality. Tanzania is the 5th country in the world with the highest births prevalence of SCD individuals. Significant advances in the neonatal diagnosis of SCD using rapid point-of-care testing have been made. However genetic confirmation is still required for positive cases, in uncertain cases, in multiply transfused patients, to resolve compound heterozygosity (Hb S/ β0 Thal or Hb S/ β+ thal) not uncommon in the coastal regions of East Africa and increasingly also for pre-marital counselling and potentially for future curative approaches such as gene therapy. The currently available DNA tests are prohibitively expensive. Here, we describe an easy-to-use, affordable and accurate β-globin sequencing approach that can be easily integrated within existing NBS for SCD and other haemoglobinopathies especially in Low- and Middle-income Countries. Aim To evaluate an affordable DNA technology for the diagnosis of Sickle cell disease and other haemoglobinopathies in a resource-limited setting. Methods Laboratory-based validation study was conducted by Muhimbili University of Health and Allied Sciences and the University of Oxford involving sequencing of the entire β -haemoglobin locus using the Oxford Nanopore MinION platform. A total number of 36 Dried blood spots and whole blood samples were subjected to conventional protein-based methods (isoelectric focusing, HPLC), and/or sequenced by the Sanger method as comparators. Results Sequencing results for SCD using the MinION were 100% concordant with those from the Sanger method. In addition, the long-read DNA sequencing method enabled the resolution of cases with unusual phenotypes which make up 1% of all children in Tanzania. The cost is £11/ sample for consumables, which is cheaper compared to other sequencing platforms. Conclusions This is the first report of a comprehensive single DNA assay as a definitive diagnostic test for SCD and other haemoglobinopathies. The test is fast, precise, accurate and affordable.

Intermittent Hypoxia Exposure Helps to Restore the Reduced Hemoglobin Concentration During Intense Exercise Training in Trained Swimmers

In prolonged intense exercise training, the training load of athletes may be reduced once their hemoglobin concentrations ([Hb]s) are decreased dramatically. We previously reported that intermittent hypoxia exposure (IHE) could be used to alleviate the decrease of [Hb] and help to maintain the training load in rats. To further explore the feasibility of applying IHE intervention to athletes during prolonged intense exercise training, 6 trained swimmers were recruited to conduct a 4-week IHE intervention at the intervals after their [Hb] dropped for 10% or more during their training season. IHE intervention lasted 1 h and took place once a day and five times a week. Hematological and hormonal parameters, including [Hb], red blood cells (RBC), hematocrit (Hct), reticulocytes, serum erythropoietin (EPO), testosterone (T) and cortisol (C) were examined. After the IHE intervention was launched, [Hb], RBC and Hct of the subjects were increased progressively with their maximum levels (P < 0.01) showing at the third or fourth week, respectively. An increase in reticulocyte count (P < 0.01) suggests that IHE intervention promotes erythropoiesis to increase [Hb]. Besides, serum level of EPO, the hormone known to stimulate erythropoiesis, was overall higher than that before the IHE intervention, although it was statistically insignificant. Furthermore, the serum level of T, another hormone known to stimulate erythropoiesis, was increased progressively with the maximum level showing at the fourth week. Collectively, this study further confirms that IHE intervention may be used as a new strategy to prevent intense exercise training-induced reductions in [Hb].

Adding Hydroxyurea to Chronic Transfusion for Sickle Cell Anemia Reduces Transfusion Burden: Final Results of the HAT Prospective Trial

Background: Chronic red blood cell (RBC) transfusion is an established therapy to prevent stroke in patients with sickle cell anemia (SCA). Chronic transfusion, however, requires considerable blood resources and has risks so a strategy to decrease transfusion requirements is highly desirable especially given blood shortages. Chronic transfusion also can fail to prevent progressive cerebrovascular disease so further optimization of this therapy is needed. Hydroxyurea has been studied as an alternative to chronic transfusion, but alone it provides inadequate cerebrovascular protection for patients at highest risk for stroke. Combination therapy hydroxyurea and transfusion (HAT) may provide benefits over either therapy alone because each ameliorates the pathology of SCA via different mechanisms. To evaluate the feasibility and impact of HAT on transfusion requirements, we conducted a single-arm clinical trial (NCT03644953). Methods: Pediatric patients with SCA already receiving simple chronic transfusion for stroke prevention for at least one year were approached. Patients with poor adherence to chronic transfusion defined by having a hemoglobin (Hb) S >45% and a transfusion interval >5 weeks in the last year were excluded. Enrolled patients were started on hydroxyurea 20 mg/kg/day and continued on simple chronic transfusions with the same transfusion protocol. Transfusion volumes were based on the pre-transfusion Hb to achieve a target post-transfusion Hb of 11.5-12.0 g/dL. Hydroxyurea was dose escalated to achieve a HAT target dose (HAT-TD) defined by either an absolute neutrophil count <4000/µL, or a pre-transfusion Hb >10g/dL with HbS >30%. Transfusion volumes and pre-transfusion laboratory values were compared the year before HAT and the year after HAT-TD. Results: A total of 19 eligible patients were offered participation and 14 enrolled (74% recruitment ratio). The median age was 11.2 years (range 5.3-19.8). The primary indication for chronic transfusion was history of an abnormal transcranial Doppler ultrasound (n=7), overt stroke (n=6), and progression of cerebral vasculopathy (n=1). Five participants ended the study prematurely (care transferred to another hospital, nonadherence to transfusions, patient desire to not take medications, hair thinning, family concern after a hospitalization); none had a serious adverse event that was attributed to HAT. Among the 9 participants who completed the study, the median HAT-TD was 25 mg/kg/dose (range 20-30) and was achieved after a median of 18 weeks (range 0-26). The median hydroxyurea adherence ratio, measured by pill counts, was 91%. One patient with baseline splenomegaly developed splenic sequestration requiring hospitalization; there were no other serious adverse events attributed to HAT. One patient developed a pre-transfusion Hb >11 g/dL and HbS >45%. This patient had hydroxyurea held for 12 weeks and was changed to partial manual exchange transfusions. With HAT, patients had a significant increase in pre-transfusion total Hb, HbS, HbF, and MCV; and a significant decrease in ANC and LDH (Table). After HAT-TD, 8/9 participants received fewer RBCs with a significant median RBC volume reduction of 19.4 ml/kg/year (p=.02, Figure). The single patient who received more RBCs after HAT-TD had suspected nonadherence to hydroxyurea returning only 33% of dispensed hydroxyurea bottles. While receiving HAT 8/9 patients had brain magnetic resonance imaging for clinical monitoring after a median of 75 weeks (range 29-100), no patient had a new infarct or progressive vasculopathy. Conclusion: Hydroxyurea added to chronic transfusion therapy for patients with SCA is feasible. HAT effectively decreases transfusion volumes so can help conserve blood resources. While Hb S% increases with HAT, this increase is relatively small and markers of hemolysis improved. Most patients are able to safely continue simple transfusions when hydroxyurea is added, but a few may require exchange transfusions. The effect of HAT on cerebrovascular disease warrants further study. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Hemoglobin Variants Influence Plasmodium Falciparum Sexual Differentiation

It has long been recognized that individuals who express variations of the hemoglobin-A (HbA) protein experience less severe malaria disease. As malaria remains to be one of the most significant infectious diseases in history, this human adaptation has led to the persistence of HbA variants (HbVARs) in the population. The intricate lifecycle of the parasite which causes the most cases of clinical malaria, Plasmodium falciparum, relies on both asexual and sexual reproductive cycles, with host to vector transmission reliant on sexual stage gametocyte formation. Multiple epidemiological studies have shown that HbVARs may influence gametocyte production during P. falciparum infection, with greater gametocyte numbers reported in individuals with hemoglobin variant containing erythrocytes (Hb VAR-Ery) when compared to hemoglobin A containing erythrocytes (Hb A-Ery). Here we provide experimental support for these studies by showing significantly higher sexual differentiation rates among parasites grown in Hb S containing erythrocytes (Hb S-Ery) obtained from sickle cell patients than those differentiated in Hb A-Ery (p=0.038). Because the digestion of hemoglobin is such an integral part of the intraerythrocytic cycle, we then sought to determine whether there was a difference between the hydrolysis efficiencies of HbA and other hemoglobin variants (HbVAR). By using a prominent recombinant P. falciparum hemoglobinase we found the hydrolysis efficiency of HbA to be significantly (p=0.0058) more efficient after 24 hours compared to a HbVAR sample containing mixed amounts of HbA, HbF, and HbS. To further determine whether there is a link between hemoglobin digestion efficiency and sexual differentiation, we therapeutically inhibited the hemoglobin digestion and hemozoin formation process in a culture of P. falciparum using sub-optimal doses of chloroquine diphosphate. We found a significant difference (p<0.001) among gametocyte conversion rates between treated and non-treated cultures, as well as a moderate negative correlation between hemozoin formation and gametocyte conversion rate (Pearson r=0.72, p=0.008). Gene expression analysis also revealed patterns of expression that were consistent with increased gametocytogenesis. We conclude that hemoglobin type plays a significant role in the process of sexual conversion in P. falciparum. Though further studies should be completed in order to confirm these results, these findings may suggest hemoglobin digestion efficiency as a causative factor for sexual differentiation. As individuals with hemoglobinopathies make up approximately 7% of the global population, and malaria infection rates have been shown to differ depending on these genetic dynamics, these findings may support the creation of targeted initiatives to reduce transmission specifically in areas where there is a high percentage of hemoglobinopathy carriage. Disclosures No relevant conflicts of interest to declare.

Clinical & Laboratory Profile of Possible Sickle Delta Beta Thalassemia in Telangana State

BACKGROUND Sickle delta beta thalassemia is a rare genetic disorder, with varied symptoms, signs, requiring careful monitoring for potential complications. It is due to sickle mutation and thalassemia mutation occurring together, with sparse data available worldwide. The purpose of this study was to assess the clinical and laboratory profile of possible sickle delta beta thalassemia. METHODS The study design is retrospective analysis of clinical information of those selected patients done in our multi-specialty tertiary care referral hospital situated in Telangana state in south India. The case material was collected from December 2017 to December 2019 (2 years duration). All haemoglobin electrophoresis reports were collected with no prior blood transfusions in preceding 4 months. The information collected was analysed and presented. RESULTS Total 9 patients were diagnosed as possible sickle delta beta thalassemia, with male to female ratio of 5 : 4 and age ranging from 12 years to 45 years of age. The commonest symptoms were joint pain and jaundice in 5 patients and sign was splenomegaly in 2 patients. Ultrasonogram of abdomen showed that 3 patients had gall stones, 1 patient had gall bladder sludge, 1 patient had autosplenectomy and 3 patients had splenomegaly. Mild to moderate anaemia was seen with reticulocytosis, sickling test positive in all patients, with haemoglobin in the range of 5.5 g/dl to 12.7 g/dl. 3 patients had iron over load, 2 patients had hepatopathy, 5 patients had unconjugated hyperbilirubinemia, Acute chest syndrome, hepatic necrosis, and nephropathy was seen in 1 patient each. Haemoglobin electrophoresis showed Hb S was from 46.1 % to 76.4, Hb A from 5.3 % to 34.7 %, Hb F from 4.8 % to 22.7 %, Hb A2 from 1.5 % to 3.3 %. 2 patients were treated with hydroxyurea. 2 patients had mutation analysis elsewhere that was reported as compound heterozygous for β-globulin gene for Hb S (GAG-GTG) and IVS 1 - 5 (G-C). CONCLUSIONS Sickle delta beta thalassemia presents as mild to moderate anaemia haemolysis, splenomegaly with vaso-occlusive crises. Hydroxyurea may help in the treatment. Genetic analysis helps in diagnosis and future therapies. KEYWORDS Haemoglobin S, Haemoglobin A2, Haemoglobin A, Haemoglobin F

Multispectral Imaging for MicroChip Electrophoresis Enables Point-of-Care Newborn Hemoglobin Variant Screening

Hemoglobin (Hb) disorders affect nearly 7% of the world's population. Globally, around 400,000 babies are born annually with sickle cell disease (SCD), primarily in sub-Saharan Africa where morbidity and mortality rates are high. Although treatments are available for Hb disorders, screening, early diagnosis, and monitoring are not widely accessible due to technical challenges and cost, especially in low-and-middle-income countries. We hypothesized that multispectral imaging will allow sensitive hemoglobin variant identification in existing affordable paper-based Hb electrophoresis, which is a clinical standard test for Hb variant screening. To test this hypothesis, we developed the first integrated point-of-care multispectral Hb variant test: Gazelle-Multispectral. Here, we evaluated the accuracy of Gazelle-Multispectral for Hb variant newborn screening in 321 completed tests in subjects younger than 6 months with known hemoglobin variants including hemoglobin A (Hb A), hemoglobin F (Hb F), hemoglobin S (Hb S) and hemoglobin C (Hb C). Gazelle-multispectral detected levels of Hb A, Hb F, Hb S, and Hb C, demonstrated high correlations with the results reported by laboratory gold standard high performance liquid chromatography (HPLC) at Pearson Correlation Coefficient = 0.97, 0.97, 0.89, and 0.94. Gazelle-multispectral demonstrated 100% sensitivity and 100% specificity in both disease vs normal and disease vs trait, 98.1% sensitivity and 97.0% specificity in trait vs normal in comparison to HPLC in newborns. The ability to obtain rapid and accurate results on newborn samples suggest that Gazelle-Multispectral is suitable for large-scale newborn screening and potentially for accurate diagnosis of SCD in low resource settings.

Sickle Cell Anemia: A review

La anemia hemolítica más frecuente en la población mundial es la anemia de células falciformes, con una incidencia de 1/600 recién nacidos en Estados Unidos y en España algunas regiones con incidencia de 1/5000 neonatos; en Colombia no hay registros con respecto a la incidencia y prevalencia. La transmisión de la ACF es autosómica dominante. Los homocigotos (SS), no sintetizan Hb A y poseen eritrocitos con un 90% de Hb S. El portador o heterocigoto (AS) tiene hematíes con Hb A mayor al 50% y Hb S de 20 – 40% y son usualmente asintomáticos. La Hb S se debe a una mutación en el gen de la cadena beta de globina, lo que conlleva a la polimerización de la Hb en condiciones de baja oxigenación, originándose un cambio en la morfología del eritrocito adquiriendo la forma falciforme. La sintomatología es secundaria a la anemia hemolítica crónica, la vaso–oclusión en los diferentes órganos y la asplenia funcional la cual predispone a la infección. Otras manifestaciones asociadas son el secuestro esplénico, la aplasia eritroide y las complicaciones órgano – especificas, que disminuyen la calidad de vida y predisponen a mayor mortalidad. Su manejo debe realizarse en centros de referencia donde haya un manejo integral incluyendo el recurso humano y físico, ya que el manejo inadecuado y sus complicaciones disminuyen la sobrevida la cual no es superior a los 45 años según reportes.

β-globin variants present in Western Sudan

Objective: A variety of observations of the hemoglobin D (Hgb D) phenotype has occurred in association with family studies of patients with sickle cell disease. Very little is known about the occurrence and prevalence of the Hgb D variant and its impact on blood profiles among Sudanese. This study was aimed at determining the percentage of Hb D in North Darfur State, West Sudan, whose population has been shown to have hemoglobin-S (Hb S) disease. Methods: From December 2017 to August 2018, this descriptive community-based investigation was conducted. Six hundred and sixty-six (666) people were randomly selected to participate in this study. With each participant's there was verbal consent. A questionnaire was designed to collect personal details. 5 mL of venous blood was gathered in EDTA containers. The Hb D variant was checked using Sebia Minicap Automated Capillary Electrophoresis System- USA and frequency was calculated using version 21.0 of the software package for social science (SPSS). Result: The prevalence of Hb variants was as follows: AD=0.6%, SS=2%, AS=10.5 %, and AA=86.9%. Conclusion: The prevalence of Hb D variant was 4 (0.6 %) in 666 participants from four western Sudanese tribes, beside reported of Hb SS and Hb AS.