Influence of Voxelotor–hemoglobin complexes in the estimation of hemoglobin S levels by the current standard of care laboratory evaluation techniques

BackgroundSickle cell disease is an inherited disorder characterized by the presence of sickle hemoglobin (HbS). The process of Hb molecule polymerization is a pivotal step in the sickling process. Voxelotor, a recently approved novel therapeutic agent, is known to interfere with polymerization. We aim to study the impact of Voxelotor on Hb variants analysis using high performance liquid chromatography (HPLC).Material and methodsWe are reporting the impact of Voxelotor on Hb variants analysis using HPLC after an informed consent and medical research committee approval. Data was collected from eight patients who are enrolled in the GBT440-034OL study using electronic medical records, to evaluate the Hb levels, hemolytic markers and the clinical response.ResultsOur patients were well-balanced for gender, with a mean age of 31.1 years (19–50). Six patients showed a significant improvement in the Hb level, with reduced reticulocytes, bilirubin, LDH and an improved clinical outcome. Interestingly, these patients showed the appearance of a split band of Hb S and D on HPLC impacting significantly on HbS level. Two patients did not show any improvement on laboratory parameters, and no changes on their HPLC analysis.ConclusionsWe report here eight patients on Voxelotor therapy, six of which showed improved hemolytic markers and anemia and demonstrated the appearance of HbD peak on the HPLC chromatogram. Therefore, the absence of HbD on HPLC or other laboratory methods for estimating HbS in patients on Voxelotor therapy, gives the clinician a possible hint regarding the patient's compliance with the drug

THE USE OF HPLC AS A TOOL FOR NEONATAL CORD BLOOD SCREENING OF HAEMOGLOBINOPATHY - A VALIDATION STUDY

Background: Newborn cord blood screening identifies infants with underlying haemoglobinopathies before they develop the characteristic symptoms or sequelae.  Aims: This study was performed to validate the interpretation high-performance chromatography (HPLC) along with complete blood count (CBC) results as a tool for universal neonatal screening of hemoglobin disorders in Oman.  Methods: HPLC and CBC data on subjects who participated in the National Neonatal screening program at birth were obtained from archival records. The results recorded at birth were compared with a second study performed on the same subjects, after approval from the local medical research and ethics committee. Results: Only 290 subjects from amongst the original cohort of 3740 newborns could be recalled between April 2010 to March 2011, to repeat HPLC and CBC, as well as perform confirmatory DNA studies, wherever necessary. All these subjects had been documented to show an initial abnormal result. 31 cases who had no HbA at birth on HPLC were confirmed as either homozygous β-thalassaemia major (n=5 subjects) or homozygous sickle cell anemia (n=26 subjects) by appropriate DNA analysis. Additionally, amongst 151 subjects, 72 subjects were studied in the initial study by Hb Bart’s quantitation using aalpha thalassaemia short program at birth. In this cohort, 42 subjects with Hb Bart’s >1% at birth could be confirmed as having either deletional or non-deletional thalassaemia by GAP PCR studies. No case of HbH was detected in this cohort. Further, carrier status for structural hemoglobin variants (HbS, HbC, HbD, HbE) (n=67) and beta thalassaemia allele with low HbA at birth (n=29 out of 41) were confirmed by relevant molecular studies. Conclusions: The study validated the earlier observation by 100% concordance with results of CBC and HPLC. Presence of Hb Bart’s at birth does not always mean the presence of alpha thalassemia, as subjects with Hb Bart’s was below 1% by quantitation, were shown to be normal by molecular studies.   Key Words: Neonatal, screening, HPLC validation, haemoglobinopathy, sickle cell disease, thalassaemi

A Novel Mutation (Q694X) in the ITGB3 Gene Causing Glanzmann’s Thrombasthenia from the Sultanate of Oman

Background: Glanzmann Thrombasthenia (GT) results from mutations in the genes ITGA2B and ITGB3, located on chromosome 17q21–23 which encodes the platelet glycoprotein αIIbβ3 complex, namely GPIIb (αIIb) and GPIIIa (β3), the fibrinogen receptors on platelets, which play an important role in platelet aggregation. Patients with GT can require frequent hospitalization and can be a burden on the nation’s health resources. The possibility that GT could be cured by gene replacement therapy makes it essential to study the molecular basis of the GT patients in a particular family or kindred. Objectives: Our aim was to identify the underlying mutations responsible for GT in Omani patients in order to establish a strategy for genetic counseling and carrier detection to prevent the occurrence of the homozygous state by prenatal diagnosis. Methods: GT was diagnosed in a 17 year old Omani female at the Sultan Qaboos University Hospital. The diagnosis of GT was based on clinical features, platelet aggregometry and biochemical studies. Platelet surface expression of GPIIb/IIIa was also studied by flowcytometry. Molecular studies performed at Medical Genetics Department, Tsukuba University, Japan, include DNA sequencing of all exons and exon-intron junctions of ITGA2B and ITGB3 of the two genes by the ABI 3100 Genetic Analyzer®. [Applied Biosystems, Foster City, CA, USA]. Genomic DNA was also analyzed by Illumina Human-1 Bead Chip Illumina® (Illumina Inc., San Diego, CA, USA) to exclude the whole region of the two genes that could produce an apparent homozygous state. Results: We have identified a novel nonsense causative mutation (Q694X) by sequencing the ITGB3 gene. [Figure 1a & b]. In addition, sequencing ITGB3 gene also revealed 2 SNPs (rs 3809863; IVS14+9C/T, rs 3809865; 3383T/A). The Micro-Array assay using Illumina Human-1 Bead chip excluded the possibility of deletion of these genes in chromosome 17 in this patient. Summary/Conclusion: A stop codon was found in exon 13 of ITGB3 gene causing the translated protein to be abnormally shortened. It is hypothesized that the altered form of ITGB3 gene is both extremely unstable and rapidly degraded after its biosynthesis, leading to a loss of function of the protein. Further RNA expression studies, transfection tests and cDNA sequencing are ongoing to elucidate the molecular mechanisms responsible for GT

Predicting risk factors for thromboembolic complications in patients with sickle cell anaemia - lessons learned for prophylaxis

Objective: To assess the clinical and laboratory predictors of venous thromboembolism (VTE) in patients with sickle cell anaemia (SCA) and its relationship to morbidity and mortality. Methods: This retrospective case-control study analysed data from patients with SCA that experienced VTE compared with matched control patients with SCA but no VTE (2:1 ratio). Results: A total of 102 patients with SCA were enrolled (68 cases with VTE and 34 controls). Amongst the 68 cases (median age, 29.5 years), 26 (38.2%) presented with isolated pulmonary embolism (PE). A higher prevalence of splenectomy (73.5% versus 35.3%) was observed in the cases compared with the controls. A significantly higher prevalence of central venous catheter (CVC) insertion (42.6% versus 8.8%) was observed in the cases compared with the controls. High white blood cell counts, serum lactic dehydrogenase (LDH), bilirubin and C-reactive protein (CRP) and low haemoglobin (Hb) and HbF were significant risk factors for VTE. Forty-two cases (61.8%) developed acute chest syndrome, 10 (14.7%) had a stroke and seven (10.3%) died. Conclusions: VTE in patients with SCA has a high impact on morbidity and mortality. PE was the leading presentation of VTE, with CVC insertion, high LDH, bilirubin, CRP and white blood cell counts along with low Hb and HbF constituting other significant risk factors

Nutritional and hematological status of Sudanese women of childbearing age with steady-state sickle cell anemia

We sought to investigate the nutritional and hematological status of Sudanese women of childbearing age with sickle cell anemia (SCA). Anthropometry and hematology were used to assess nutritional status and health and disease conditions, respectively. Women with steady-state (HbSS, n = 39; age = 19.0±2.7) and without (HbAA, n = 36; age, 19.8±2.7) SCA were recruited during a routine visit to the Hematology Clinic, Ibn-Auf Teaching Hospital, Khartoum, Sudan. The two groups of women lived in similar environmental conditions and ate similar diets three times a day. However, despite taking regular meals, the women with sickle anemia were thinner and lighter ( 0.050). The low anthropometric (height, weight, and body mass index) and abnormal hematological values in the women with SCA in steady-state reflect sustained nutritional insults inflected by the disease and poverty. Tailored nutritional counseling/advice must be an integral part of managing patients with SCA. Such advice is particularly vital for women of childbearing age because of the adverse effects of prepregnancy nutritional deficiency on outcomes

Artificial intelligence in sickle disease

Artificial intelligence (AI) is rapidly becoming an established arm in medical sciences and clinical practice in numerous medical fields. Its implications have been rising and are being widely used in research, diagnostics, and treatment options for many pathologies, including sickle cell disease (SCD). AI has started new ways to improve risk stratification and diagnosing SCD complications early, allowing rapid intervention and reallocation of resources to high-risk patients. We reviewed the literature for established and new AI applications that may enhance management of SCD through advancements in diagnosing SCD and its complications, risk stratification, and the effect of AI in establishing an individualized approach in managing SCD patients in the future. Aim: to review the benefits and drawbacks of resources utilizing AI in clinical practice for improving the management for SCD cases.Open Access funding provided by the Qatar National Library.Scopu