HbA2 Levels Research Articles

Common mutation analysis of beta-thalassemia carriers by amplification refractory mutation system-polymerase chain reaction in cases diagnosed by capillary zone electrophoresis at National Public Health Laboratory, Nepal

Abstract BACKGROUND: Beta (β)-thalassemia is a globally prevalent genetic disorder with over 350 known mutations in the β-globin gene. Intervening-sequence (IVS) 1–5 (G→C), 619 bp deletion, IVS 1–1 (G →T), C-15 (G→A), frameshift (FS) 41/42 (−CTTT), FS 8/9 (+G), codon 19, A-G, and codon 17 (A→T) are some common mutations in β-thalassemia. This study aimed to identify and analyze the common mutation by amplification refractory mutation system polymerase chain reaction in β-thalassemia carriers diagnosed by capillary zone electrophoresis (CZE) at National Public Health Laboratory. MATERIALS AND METHODS: On the basis of archived data, the CZE performed on 781 samples between June 2019 and July 2022 was analyzed. Without regard to age, gender, race, or address, 121 samples were ultimately selected for mutation analysis during this time because they were identified as positive for the β-thalassemia traits based on high HbA2 levels of more than 3.5%. RESULTS: IVS 1–5 (G→C) was the most common mutation followed by 619 bp deletion. Five mutations namely IVS 1–5 (G→C), 619 bp deletion, FS 41/42 (−TTCT), FS 8/9 (+G), and codon 15 (G→A) accounted for 84.3% of cases. A single mutation was seen in 87 cases (71.9%), more than one mutation in 15 cases (12.4%) whereas 19 cases (15.7%) did not show any of the mutations tested. CONCLUSION: IVS 1–5 (G-C) is the most prevalent mutation in Southeast Asian nations, with the 619 bp deletion predominant in the Tharu community. Further research is needed to understand diverse ethnic groups and regions, enabling prenatal diagnostics and genetic counseling for β-thalassemia prevention.

Genetic epidemiology of thalassemia in couples of childbearing age: over 6 years of a thalassemia intervention project.

Shenzhen is one of the most populated metropolises in southern China where thalassemia is highly prevalent. The prevention of thalassemia inheritance is an ambition of child-bearing couples. A total of 22,098 peripheral blood samples were collected from 11,049 potentially at-risk couples of childbearing age from Shenzhen. Thalassemia mutations were determined by PCR-based flow-through hybridization. The results identified 45.02% of the participants (9948 out of 22,098) as harboring globin gene mutations, distributed into 18 α-thalassemia alleles detected in 71.48% (7111 out of 9948) and 15 β-thalassemia alleles detected in 32.68% (3252 out of 9948) of all mutant individuals, among which 415 individuals carried both α- and β-thalassemia alleles. The most frequent phenotypes for α-globin variations were --SEA/αα (63.37%), -α3.7/αα (18.66%), and -α4.2/αα (7.31%), and those for β-globin variations were β41-42/βN (34.96%), β654/βN (28.11%), and β17/βN (13.84%). A total of 970 high-risk couples who could possibly give birth to offspring with thalassemia intermedia or major were identified. In addition, the hematological indices were compared among thalassemia genotypes. Significant differences in MCH, MCV, Hb A, and Hb A2 levels among α-thalassemia minor (α+), trait (α0), and intermediate phenotypes (P < 0.05) and between βE/βN and the other β-thalassemia phenotypes (P < 0.05) were found. Moreover, GAP-PCR and next-generation sequencing further identified 42 rare mutations, 13 of which were first reported in the Chinese population. A novel mutation in the β-globin gene (HBB: c.246C > A (rs145669504)) was also discovered. This study presented a comprehensive analysis of thalassemia variations in a population from Shenzhen and may offer valuable insights for thalassemia control and intervention strategies in this area.

A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F.

Hemoglobinopathies, including thalassemia and hemoglobin (Hb) variants, are common hematological disorders in tropical countries. Accurate and precise separation of hemoglobin types and reliable quantitation are necessary for differential diagnosis of these disorders. We have evaluated the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC; Trinity Biotech, Co. Wicklow, Ireland) to assist in the presumptive diagnosis of thalassemia and Hb variants commonly found in Southeast Asian countries. HbA0, HbA2, HbE, and HbF levels were separated and quantified in 120 blood samples from unrelated adult subjects and compared with those analyzed by capillary zone electrophoresis (CZE; CAPILLARYS™ 2, Sebia, Norcross, GA, US). The Hb analysis patterns of Hb variants obtained from the PR-HPLC system were also compared to those obtained from HPLC (VARIANT II, β-thalassemia Short Program, Bio-Rad, Laboratories, Hercules, CA, US) and CZE systems. The PR-HPLC had excellent precision with a coefficient of variation (CV) for HbA2 quantitation of 3.8 % within-run and 5.2 % between-run. The levels of HbA2/E quantified by the PR-HPLC system correlated well with those of the CZE system (r=0.997). In addition, thalassemia interpretation results obtained from the PR-HPLC and the CZE showed 100 % agreement. Moreover, chromatograms of the PR-HPLC were also comparable to those of VII-HPLC and CAP2-CZE electropherograms. The PR-HPLC system would be applicable to diagnose common forms of thalassemia and Hb variants in Southeast Asia.

Third-generation sequencing identified two rare α-chain variants leading to hemoglobin variants in Chinese population.

Rare and novel variants of HBA1/2 and HBB genes resulting in thalassemia and hemoglobin (Hb) variants have been increasingly identified. Our goal was to identify two rare Hb variants in Chinese population using third-generation sequencing (TGS) technology. Enrolled in this study were two Chinese families from Fujian Province. Hematological screening was conducted using routine blood analysis and Hb capillary electrophoresis analysis. Routine thalassemia gene testing was carried out to detect the common mutations of α- and β-thalassemia in Chinese population. Rare or novel α- and β-globin gene variants were further investigated by TGS. The proband of family 1 was a female aged 32, with decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Hb A2, and abnormal Hb bands in zone 5 and zone 12. No common thalassemia mutations were detected by routine thalassemia analysis, while a rare α-globin gene variant Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] was identified by TGS. Subsequent pedigree analysis showed that the proband's son also harbored the Hb Jilin variant with slightly low levels of MCH, Hb A2, and abnormal Hb bands. The proband of family 2 was a male at 41 years of age, exhibiting normal MCV and MCH, but a low level of Hb A2 and an abnormal Hb band in zone 12 without any common α- and β-thalassemia mutations. The subsequent TGS detection demonstrated a rare Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant in HBA2 gene. In this study, for the first time, we present two rare Hb variants of Hb Jilin and Hb Beijing in Fujian Province, Southeast China, using TGS technology.

Two cases of vitamin B12 deficiency in patients with beta-thalassemia trait: lessons in diagnosis

BackgroundCoexisting pathologies is not a new phenomenon and can cause diagnostic difficulties when they overshadow one another. They may alter the cytomorphology of cells and the machine readings to an extent that the actual diagnosis is missed. One such scenario is created by coexisting megaloblastic anemia and beta-thalassemia trait where the RBC indices and the hemoglobin chromatogram findings conform to neither of the two. Case presentationWe present two cases of anemia presenting with normocytic normochromic indices, increased red cell distribution width (RDW), elevated HbA2 levels and moderately high HbF levels in both of them and an additional low HbA1c in case 2. Upon evaluation of peripheral smear and biochemical tests, a non-immune hemolytic component was also identified with deficient Vit B12 levels in case 1 and highly elevated levels post-supplementation in case 2. ConclusionsTaking together the peripheral smear, erythrogram, chromatogram and biochemical findings, it was deduced that the alterations were caused by coexisting beta-thalassemia trait and megaloblastic anemia. Vit B12 deficiency was the cause behind hemolysis owing to intramedullary destruction of abnormal precursors. Upon supplementation in case 2, the immediate reticulocyte response was the reason behind spuriously low HbA1c.

Effect of Iron replacement therapy in Pregnant Beta Thalassemia Carrier Patients with Coexisting Iron Deficiency Anemia.

Iron deficiency anemia (IDA) and β -Thalassemia trait are two prevalent etiologies of microcytic hypochromic anemia. Pregnant women with IDA face significant risk of short-term and long-term complications for mother and newborns. The incidence of concurrent thalassaemia trait and iron deficiency was reported to be quite high but despite the high prevalence of these two conditions, the diagnosis is often underestimated and not properly managed.&#x0D; OBJECTIVE; The present study was conducted to evaluate impact of iron therapy in pregnant beta thalassemia minor patients with iron deficiency anemia.&#x0D; METHODS; Study Group constitutes 60 beta thalassemia trait pregnant women with iron deficiency anemia (IDA) with Hb less than 11g/dl, HbA2 levels &gt;3.5% on HPLC and low serum ferritin (&lt;30 ng/mL).After the initial investigations, participants received iron supplementation as 60 mg elemental iron three times daily for duration of 8 weeks. After completing therapy, complete blood count and serum ferritin were performed.&#x0D; RESULTS; Iron replacement therapy results in effective increment in hemoglobin, red cell indices and Serum ferritin.&#x0D; CONCLUSION; Pregnant women with beta-thalassemia minor combined with IDA can receive Iron replacement therapy for improvement in Hemoglobin effectively.

Reliability of hemoglobin A2 value as measured by the Premier Resolution system for screening of β-thalassemia carriers.

Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing β-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE). We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA2 cutoff value for β-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the β- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias,-0.33; r, 0.991), β-thalassemia (bias,-0.27; r, 0.927), and non-β-thalassemia carriers (bias,-0.36; r, 0.928). An HbA2 cutoff value of≥4.0 % for β-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare β-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with β-thalassemia and co-inherited with a single α-globin gene. The Premier Resolution HPLC is a reliable and accurate method for routine β-thalassemia carrier screening, aligning with existing CE methods.

Molecular understanding of unusual HbE-β+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels

Background Low HbF expression in HbE-β+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the β- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-β+-thalassemia from HbE heterozygotes have been investigated. Participants and Methods A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize β- and α-globin genes. DNA polymorphism at position −158 nt 5′ to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-β+-thalassemia from HbE heterozygotes were determined. Results Five β+-thalassemia mutations trans to βE-gene (β−87(C>A), β−31(A>G), β−28(A>G), β19(A>G), and β126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000–1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden’s index for differentiating HbE-β+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-β+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at −158 nt 5′ to Gγ-globin was found to be negatively associated with HbF expression. Conclusions HbE-β+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-β+-thalassemia diagnosis.

Molecular analysis and clinical significance of hemoglobin Quong Sze in Huizhou city, Southern China.

Hemoglobin Quong Sze (Hb QS) is one of the most common non-deletional α-thalassemia (α-thal), which is prevalent in the Southern Chinese population. However, there are still few comprehensive researches on the molecular characterization of Hb QS. So it is important to find out appropriate diagnosis and characterization of Hb QS carrier for genetic counseling. A hematological screening including hematological indices and hemoglobin analysis was performed in 113,400 individuals from Huizhou city, Southern China. Then, suspected thalassemia carriers were detected by a suspension-array system and DNA sequencing for α- and β-thal. In our study, we identified 521 subjects who were Hb QS carriers, including fourteen different genotypes. Among them, 445 Hb QS heterozygotes showed a decrease in the mean corpuscular hemoglobin (MCH), 16 compound heterozygotes for Hb QS/α+-thal presented mild thalassemia, 28 Hb QS in combination with --SEA/αα manifested as Hb H disease, varying clinical symptoms from only moderate anemia to severe anemia and requiring blood transfusion, and 29 double heterozygotes for Hb QS and β-thal behaved as β-thal trait. The mean corpuscular volume (MCV) and MCH were significantly reduced and no Hb H peak could be detected in one patient with Hb H-Hb QS and β-thal. Meanwhile, we identified two homozygous Hb QS carriers, who showed mild to moderate anemia and increased Hb A2 level but negative results from a sequencing analysis for the first time. Additionally, Comparison of hematological parameters among the major four genotype groups showed significant differences in most box-whisker plots. People who originated from Huizhou city showed many genotypes and diversity in the clinical manifestations of Hb QS carriers. This study enlarges the mutation spectrum of α-thal and emphasizes that reliable detection of the gene mutations is important for genetic counseling. It also strengthens the prevention and control of thalassemia.

Mutational spectrum of HBD gene in the Chinese population: Description of 36 mutations including 11 novel variants.

Mutations in the hemoglobin subunit delta (HBD) gene (MIM#142000) are associated with decreased levels of the Hemoglobin A2 (Hb A2 ) fraction. We aimed to examine the prevalence of HBD gene mutations and summarize their characteristics in the Chinese population. Individuals who exhibited Hb A2 levels below 1.8%, with or without Hb A2 variant peaks, were chosen for further investigation. Hemoglobin analysis was conducted using capillary electrophoresis. Common α and β-thalassemia in China were detected using gap-PCR and reverse dot blot hybridization. The presence of HBD gene mutations was confirmed by DNA sequencing. A total of 188 patients were identified as carriers of the HBD gene mutation, with a prevalence of approximately 0.46%. We discovered 36 types of mutations, 30 of which resulted in δ-globin variants, while the remaining 6 resulted in δ-thalassemia. The most common mutation was HBD:c.-127 T > C, accounting for 87.2% of δ-thalassemia cases. In addition, we identified 11 novel HBD gene mutations and found 10 cases compounded with other common thalassemias. We observed a high prevalence of HBD gene mutations in southern China. Our findings provide a genetic basis for screening for δ-thalassemia and enrich the spectrum of HBD gene mutations.

Effective screening of hemoglobin Constant Spring and hemoglobin Paksé with several forms of α- and β-thalassemia in an area with a high prevalence and heterogeneity of thalassemia using capillary electrophoresis

Background and aimsWe aimed to evaluate the efficiency of identification and quantification of hemoglobin (Hb) Constant Spring (CS) and Hb Paksé by capillary electrophoresis (CE). Materials and methodsBlood samples collected from 2057 patients were used for identifying and quantifying Hb by CE. Molecular analysis of α- and β-thalassemia, Hb CS, and Hb Paksé was performed. ResultsHb CS and Hb Paksé were identified in 573 samples (27.86%) with diverse genotypes. Thirty-eight samples (6.6%) showed no Hb CS peak. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of Hb CS by CE were 93.37, 95.96, 89.92, 97.40, and 95.24%, respectively. The amount of Hb CS in those carrying Hb CS was 0.2–6.5% which showed an increasing trend according to the number of defective α-globin genes, in contrast to Hb A2 levels, which decreased. Hb CS level ≥1.0% accurately excluded heterozygotes and that of ≥2.0% could identify homozygotes. ConclusionCE has the high potential for identifying and quantifying Hb CS and Hb Paksé, especially in an area with a high prevalence of thalassemia. Hb CS levels can be used as a potential marker to distinguish the genotype of individuals carrying Hb CS.

Evaluation of Effect of Iron Deficiency Anemia on HbA2 Levels

Objective: To evaluate the effect of iron deficiency anemia on HbA2 levels. Study Design: Comparative cross-sectional study. Place and Duration of Study: Department of Pathology, Combined Military Hospital, Lahore Pakistan, from Jan to Oct 2020. Methodology: A total of 206 subjects were included in the study out of which 56 served as healthy controls. Five milliliters of whole blood were extracted in an EDTA tube and basic haematological parameters were performed using automated hematology analyzer (Sysmex KX-21). HbA2 levels were determined by cellulose acetate hemoglobin electrophoresis at alkaline pH (Mini Hor-8). Serum Ferritin levels were measured by chemiluminescence on Cobas e411 and Serum Iron levels by spectrophotometric method using ferrozine (Cobas c501). Results: Mean hemoglobin in control group was 12.5±0.7 g/dL and in iron deficiency anemia group was 8.1±1.36 g/dL (p=0.01). Mean HbA2 level in control group was 2.69±0.24% and iron deficiency anemia group was 1.76±0.25 % (p=0.001). HbA2 levels were reduced in individuals having haemoglobin levels less than 10g/dL and remained unchanged/minimally changed with hemoglobin levels reaching near normal levels. Conclusion: There was a statistically significant association of HbA2 levels with degree of anemia and serum ferritin levels (p=0.001).

Comparison of HbA2 Using High Performance Liquid Chromatography Versus Haemoglobin Capillary Zone Electrophoresis.

Thalassemia is among the most common hereditary disorders in the world. Approximately 5% of the world's population are carriers of hemoglobinopathies, and 2.9% are carriers of beta thalassemia. Haemoglobin A2 (HbA2) constitutes less than 3% of the total hemoglobin (Hb) in adults, and the determination of Hb A2 levels is important to diagnose the beta thalassemia trait(BTT). In some cases, the level of HbA2 is not typically elevated, and some difficulties may arise in making the diagnosis. Cation exchange high-performance liquid chromatography (HPLC) and HbCZE (haemoglobin capillary zone electrophoresis) are considered acceptable methods to diagnose BTT, but these vary in their accuracy and cut-offs. In this study, we attempted to compare HbA2 values using two methods, HPLC and HbCZE, in 536 whole blood samples sent by physician-ordered hemoglobinopathy screening over two years. This included antenatal women, patients with anemia not responding to iron, and cases of familial screening where either a child or a sibling had been diagnosed with hemoglobinopathy or thalassemia. The performance characteristics of both machines were compared for the detection of the 5 most common hemoglobin variants: Hb A, HbF, HbS, Hb C, and HbE. On comparing the HbA2 values, the HPLC showed higher values for HbA2 as compared to HbCZE, while the HbF and HbS measurement agreement was good between both methods. Normal ranges and mean normal values of HbA2 differ between different methods and different manufacturers; hence, each institute using these machines should validate its cutoffs.

Prospective screening for δ-hemoglobinopathies associated with decreased hemoglobin A2 levels or hemoglobin A2 variants: A single center experience

Backgroundδ-hemoglobinopathies may lead to misdiagnosis of several thalassemia syndromes especially β-thalassaemia carrier, it is important to evaluate the δ-globin gene defects in areas with high prevalence of globin gene disorders. We describe a prospective screening for δ-hemoglobinopathies in a routine setting in Thailand. MethodsStudy was done on a cohort of 8,471 subjects referred for thalassemia screening, 317 (3.7%) were suspected of having δ-globin gene defects due to reduced hemoglobin (Hb) A2 levels and/or appearance of Hb A2-variants on hemoglobin analysis. Hematologic and DNA analysis by PCR and related assays were carried out. ResultsDNA analysis of δ-globin gene identified seven different δ-globin mutations in 24 of 317 subjects (7.6%). Both known mutations; δ−77(T>C) (n = 3), δ−68(C>T) (n = 1), δ−44(G>A) (n = 8), Hb A2-Melbourne (n = 5), δIVSII−897(A>C) (n = 5), and Hb A2-Troodos (n = 1) and a novel mutation; the Hb A2-Roi-Et (n = 1) were identified. This Hb A2-Roi-Et, results from a double mutations in-cis, δCD82(AAG>AAT) and δCD133(GTG>ATG), was interestingly found in combination with an in trans, 12.6 kb deletional δβ0-thalassemia in an adult Thai woman who had no Hb A2 and elevated Hb F. A multiplex-allele-specific PCR was developed to detect these novel δ-globin gene defects. ConclusionsThe result confirms a diverse heterogeneity of δ-hemoglobinopathies in Thailand which should prove useful in a prevention and control program of thalassemia in the region.

DIAGNOSTIC ACCURACY OF RDWI TO DIFFERENTIATE IRON DEFICIENCY ANEMIA AND THALASSEMIA TRAIT IN BORDERLINE HBA2

Background: Thalassemia is an inherited hemoglobin disorders in which there is reduction or absence of α or β chains due to mutations in α or β genes. Microcytic hypochromic anemia is hallmark of beta thalassemia trait and the patient is usually asymptomatic. It is important to differentiate it from iron deficiency anemia (IDA) which also shows similar peripheral blood picture. The diagnosis is based on quantification of HbA2 and serum ferritin. If the level of HbA2 is more than 3. 5%, then diagnosis of beta thalassemia trait is made. The problem arises with borderline HbA2 i. e 3.1% - 3.4%. There is also diagnostic difficulty when concomitant iron deficiency anemia is present.&#x0D; Objectives: To determine the diagnostic accuracy of RDWI in borderline HbA2 to distinguish the beta thalassemia trait from IDA and to diagnose beta thalassemia trait when genetic analysis is not easily available.&#x0D; Methods: It was a cross sectional validation study. A total of 90 patients were included in this study, having HbA2 between 3.1-3.4%. Tests for serum ferritin, serum iron and total iron binding capacity (TIBC) were done on all samples. RBC morphology, RBC count, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and RDW of all samples were noted. RDWI of all samples was calculated. Mutational analysis by ARMS- PCR was done for confirmation of β thalassemia trait.&#x0D; Results: Out of 90 samples with borderline HbA2, 30 cases were diagnosed as β thalassemia trait. Out of these, 15(50 %) had concomitant IDA along with beta thalassemia trait and 15 were non iron deficient beta thalassemia carriers. 43 samples were not iron deficient and were also negative for β thalassemia trait, whereas 17 samples had only IDA without beta thalassemia trait. The sensitivity of RDWI was 76.6% and specificity was 56.6% in borderline HbA2.&#x0D; Conclusion: RDWI is a good indicator to differentiate between IDA and beta thalassemia trait (sensitivity 76.6% specificity 56.6%). But in all cases of borderline HbA2 genetic analysis should be done to see the mutation and to confirm the diagnosis.

εγ-Thalassemia, a New Hemoglobinopathy Category.

Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for β-thalassemia. Notably, ϵγδβ-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψβ loci with intact LCR, δ-, and β-regions in 2 women and newborn twins. Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψβ (HBBP1) loci. This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδβ-thalassemia.

High Performance Liquid Chromatographic Pattern of Children with Sickle Cell Anaemia in Enugu Nigeria

Aim: To identify the quantity of haemoglobin (Hb) ‘S’(HbS), ‘A2’(HbA2), ‘F’(HbF) and other Hb phenotypes of sickle cell anaemia (SCA) children in Enugu Nigeria using High Performance Liquid Chromatography(HPLC).&#x0D; Introduction: The inheritance of two abnormal Hb genes one of which must be the Hb ‘S’ gene result in Sickle cell disease (SCD). Co-inheritance of two Hb S genes results in homozygous SCD/SCA), coinheritance of Hb ‘S’ with Hb ‘C’ gene gives HbSC disease and with β-thalassemia allele gives HbSβ-thalassemia (Hb S/β Th). SCA is the most common and most severe of the SCD. The phenotypic expressions of Hb vary unpredictably in the same individual and contribute to the varied clinical severity of SCA among other variables. It is therefore imperative to characterize the Hb variants in children with SCA for proper risk stratification necessary for best outcome management. HPLC is the most validated method for screening, detection and quantification of various Hb subtypes.&#x0D; Methodology: A cross-sectional, descriptive study involving 75 SCA children aged 6 months to 17years, on follow-up at the clinic. Patients on hydroxyurea or who received blood transfusion within the previous four months were excluded. Following due ethical protocols, the D-10 HPLC machine (BIO-RAD D-10) was used to identfy the Hb phenotype in venous blood samples based on their ionic gradients and quantify them by the principle of variable absorbance. The participants’ sociodemographic data were recorded. Participants were grouped into 3 socioeconomic classes (SECs) as proposed by Oyedeji.&#x0D; Results: There were 48 females (62.7%) and 27 males (37.3%) in age range 6 months – 17 years in lower (16.0%), middle (57.3%) and upper (26.7%) SECs. Majority had HbF below 10% (46.7%), HbS above 80% (43%) and HbA2 of 4% and below (84%). No other Hb variant was identified. The proportion with HbS/HbA2/HbF levels suggestive of beta thalassemia was 16%, 25% males compared to 10.6% females. Females had higher HbF levels while males had higher HbS and HbA2 levels. However the gender differences in HbF, HbS, HbA2 and SECs did not attain statistical significance. A significant negative relationship was found between age and HbF (r = -.424, p &lt; .001) while a significant positive relationship between age and HbS (r = .287, p = .013) and between age and HbA2 (r = .265, p = .022).&#x0D; Conclusion: Irrespective of gender, high HbS and low HbF levels at direct variance (HbS) and indirect variance (HbF) with age may be found in children with SCA. Observed Hb phenotypes suggest co-existent β- thalassaemia in this subset of southeast Nigerian SCA children.

Molecular Basis and Hematologic Phenotype of Hemoglobin H Disease Combined with Two Rare β-Globin Mutations

In area where α-thalassemia and β-thalassemia are prevalent, the coinheritance of hemoglobin H disease (Hb H disease) and β-thalassemia are not uncommon and could result in complex thalassemia intermedia syndromes. In this study, we investigate the hematological and molecular characteristics of two previously undescribed cases that co-inherited Hb H disease and rare β-globin gene (HBB) mutations found in Chinese populations. Proband I was a boy with Hb H disease in association with IVS-II-5(G > C) (HBB:c0.315 + 5G > C) mutation. Proband II was a boy with a combination of Hb H and Hb Zengcheng [β114(G16) Leu > Met; HBB:c.343C > A]. Both of them had mild hypochromic microcytic anemia, and neither had ever received a blood transfusion. In both cases, the level of Hb A2 was within normal range, and no Hb H was detected, but a small amount of Hb Bart’s was observed in proband I. Routine DNA analysis detected the deletional Hb H disease in both cases. IVS-II-5(G > C) (HBB:c0.315 + 5G > C) and Hb Zengcheng (HBB:c.343C > A) mutations were found by DNA sequencing of β-globin gene. The co-inheritance of Hb H disease with rare β-thalassemia may result in an atypical pattern of Hb H disease, and further investigation of rare genotypes should be conducted to avoid missed diagnosis.

Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction – Parallel Ion Parking

β-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A2. Diagnosis of β-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/β ratio can act as a biomarker to identify β-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/β ratio as a thalassemia biomarker.

Incidence of Βeta-Thalassemia Minor among Healthy Blood Donors

Background: Disorders with a markedly slowed rate of globin chain synthesis are referred to as thalassemia. Hemoglobinopathy is a word used to describe diseases that cause structurally aberrant hemoglobin. Iron deficiency is seen in beta-thalassemia minor, which may change the typically increased HbA2 levels. According to World Health Organization (WHO) statistics, 7% of the global population carries a hemoglobin problem. Thalassemia is the most prevalent of the hemoglobinopathies, which are significant genetic issues in Pakistan. The prevalence of -thalassemia in Pakistan is around 5%, whereas the prevalence of hemoglobin S or E is between 0.5% and 1%. The purpose of the study was to determine how common beta thalassemia minor was among blood donors. Methods: A total of 500 individuals were recruited in this cross-sectional study who were referred for hemoglobin electro-phoresis between September 2015 and March 2016 were the subject of this investigation. A thorough clinical history was taken, including information on the patient's age, sex, cast, family history, history of blood transfusions, and physical findings such as splenomegaly. Hb Electrophoresis tests were performed on all blood samples. Results: Out of total 500 patients, male were 83.1% (n=79) while female were 16.9% (n=16). Among total individuals, 18.3% (n=94) were found positive cases on Hb electrophoresis technique while remaining 406 patients were observed negative. All recruited individuals were categorised in four (04) different groups based on age; highest number of individuals were found in 18-30 years of age group with 69 patients, followed by 31-40 years of age group (n=19), 41-50 years of age group (n=5.3), and 51-60 years of age group (n=01). 94 (18.3%) had abnormal hemoglobin and beta thalassemia minor. Of them, 79 (84.4%) were men and 16 (17.2%) were women. Conclusion: Many groups in Pakistan continue to struggle with hemoglobin problems. Preventive interventions, such as pre-marriage carrier status identification and screening for beta thalassemia minor, are required to lower the incidence of beta thalassemia major by forcing couples with beta thalassemia minor to abort their pregnancies. Keywords: Hemoglobin Disorders, Thalassemia, β-Thalassemia, Healthy Blood Donors, Hemoglobinopathy