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Objective: The capillary zone electrophoresis (CZE) and highperformance liquid chromatography (HPLC) methods were compared in terms of HbA2 measurement for the assessment of hemoglobinopathies. Materials and Methods: CZE was compared with HPLC for the evaluation of patients without hemoglobinopathy (n=321), with β-thalassemia trait (n=113), and with common (HbD-Punjab, E, C, S/A, and S/S) and rare (HbS/D, O-Arap, Lepore, G-Coushata, Setif, Hamadan, Q-Iran, and H) variants (n=21). The reference range for HbA2 was determined by CZE. Results: Among patients without hemoglobinopathy, the median (2.5th-97.5th percentiles) values were 97.4% (97.0-98.0%) and 97.5% (96.6-98.4%) for HbA (p=0.060) and 2.4% (1.6-3.0%) and 2.5% (1.6-3.1%) for HbA2 (p<0.001) by HPLC and CZE, respectively. The reference range for HbA2 was 1.6-3.1% by CZE. In the comparison of methods for HbA2, there was a constant error of 0.255 (confidence interval: 0.062-0.448) and bias of 0.10% (limit of agreement: 0.33- 0.53), and higher values were obtained with CZE. A strong correlation was observed between the methods (r=0.782). Interrater agreement was almost perfect for clinical diagnosis (=0.911). The two methods detected and identified the common variants similarly. All rare variants, except HbH by HPLC and HbS/D by CZE, were detected as separate peaks by both methods. Conclusion: The two methods were in agreement regarding the preliminary identification of β-thalassemia patients. Different Hb variants were detected by both methods but with possible methodological interference for HbA2 measurements. CZE is a reliable and simple alternative for the evaluation of hemoglobinopathies. The standardization of HbA2 measurements should be prioritized as more techniques become available in routine laboratory practice [ABSTRACT FROM AUTHOR]

Cation exchange high-performance liquid chromatography (HPLC) is one of the techniques available for determining glycated hemoglobin (HbA1c) and also the method of choice for structural hemoglobinopathies screening. The objective of this case is to show how in a routine HbA1c test it is possible to incidentally find a hemoglobinopathy. In a routine blood analysis, an abnormal value for the hemoglobin A2 (HbA2) was obtained during the study of HbA1c with HPLC on the ADAMS™ A1c HA-8180T. After suspecting it could be due to the presence of a hemoglobinopathy, the study of possible variants was expanded using electrophoresis and HPLC on the Hydrasys and Variant II analysers, respectively. Since it could not be identified by these conventional methods, a genetic study was also carried out using Sanger sequencing. The patient presented a low HbA2 (1.3 %) and a 24.9 % variant with a retention time of 1.95 min, compatible with alpha-globin chain variant. In the genetic study, the pathogenic variant c.138C>G was detected in the HbA2 gene in heterozygosis, which resulted in the expression of the structural hemoglobinopathy known as hemoglobin Bari. The initial screening for structural hemoglobinopathies allows its identification or suspicion especially when it was performed with HbA1c analysis, requiring subsequent confirmation and diagnosis by other techniques. [ABSTRACT FROM AUTHOR]

Haemoglobinopathies constitute a large proportion of hemolytic anemias constituting around 74% of hereditary hemolytic anemias. Till date, about 200 alpha chain variants have been identified, one of which is Hb Fontainebleau. It is a rare alpha chain variant characterized by an Alanine → Proline substitution at codon 21 with a GCT>CCT change at the molecular level. It is incidentally detected on HPLC as an unknown peak. We present a case of Hb Fontainebleau in a 53-year-old male patient who presented with symptoms related to hemolytic anemia and an unknown peak on the HPLC chromatogram. [ABSTRACT FROM AUTHOR]

Introduction: Hemoglobinopathies are major public health problems worldwide. Accurate laboratory diagnosis of the carrier is essential, which includes initial screening, Hb analysis, and DNA analysis. For the first time, we have developed a single‐tube quality control (QC) sample for these laboratory tests. Methods: The QC sample was made from a lyophilized mixture of the stabilized hemolysate with carbon monoxide saturation and the white blood cells of known thalassemia mutations. Homogeneity and stability were examined by Hb and DNA analyses on day 0 and every month for 12 months, at room temperature, 4°C, and −20°C. A preliminary proficiency testing (PT) program for hemoglobinopathies using this single QC material was developed. Results: Hemoglobin (Hb) and DNA analyses of a single‐tube QC sample demonstrated satisfactory results of Hb analysis for at least five months and DNA analysis for at least one year of storage at −20°C. The results obtained from a preliminary PT program on five expert laboratories confirmed that a single tube QC sample prepared could be used as a PT item with various Hb and DNA analyses methods. Conclusion: A single lyophilized control sample has been generated for use in hemoglobinopathies' internal and external quality control program. Unlike other available control materials, which are used for individual testing, a single‐tube QC sample generated can be used to control the pre‐analytical and analytical processes of both Hb and DNA analyses and is suitable for use in the PT program of hemoglobinopathies. [ABSTRACT FROM AUTHOR]

Background: High performance liquid chromatography (HPLC) is the most commonly used method for detection and quantitative estimation of hemoglobin variants. Hemoglobinopathies are amongst the most common genetically inherited disorders, however, the exact magnitude of different hemoglobinopathies is obscure in India. This study was done with the aim of analyzing the different findings in HPLC using D-10 analyzer and evaluating the spectrum of different hemoglobin disorders in a hospital-based population of South Delhi. Such a prevalence study would be useful to review the various strategies that can be implemented for effective control and prevention of these disorders. Methods: A hospital based descriptive observational study was conducted in which all OPD and IPD patients who were advised HPLC during their clinical workup were included. Analysis of EDTA blood samples was done by Bio Rad D10 Dual program HPLC instrument. The exact percentage of HbA, HbA2, HbF and any other variant hemoglobin was estimated. Presumptive identification of hemoglobin variants was made primarily by their percentage, retention time (RT) and peak characteristics. HPLC findings were correlated with the clinical history, family history and the CBC and peripheral smear findings in all cases. Results: On HPLC analysis, 79% of the patients had no abnormality detected and the report was within normal limits. The commonest hemoglobinopathy was Beta Thalassemia Trait followed by HbE trait. The other hemoglobinopathies detected were HbD Punjab Heterozygous (3 cases, 0.5%), Beta thalassemia homozygous (3 cases, 0.5%), Sickle cell Heterozygous (2 cases, 0.3%), HbJ Meerut Heterozygous (2 cases, 0.3%). One case each of Sickle cell Homozygous (0.15%), Compound Heterozygous HbS/beta thalassemia trait (0.15%), HbE Homozygous (0.15%), Compound Heterozygous HbE/beta thalassemia trait (0.15%), and Homozygous delta beta thalassemia (0.15%) were also diagnosed. Conclusion: This study gives an important insight to the present day scenario of hemoglobinopathies in patients in South Delhi in relation to the hematological profile. It highlights the chromatogram findings of different hemoglobinopathies on the D10 analyzer. The comprehensive data obtained by such series can help in the formulation and development of infrastructure and policies for hemoglobinopathy prevention, diagnosis and management. [ABSTRACT FROM AUTHOR]

Introduction: The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years. Materials and Methods: Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. Results: The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)0 Indian inversion and the HPFH‐3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (−α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. Conclusion: The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India. [ABSTRACT FROM AUTHOR]

Abstract: The hemoglobinopathies, as a group, are one of the most common serious monogenic diseases in the world. An accepted and widely adopted approach to reduce the number of new cases involves carrier‐screening programs, with the option of prenatal diagnosis (PND) or preimplantation diagnosis (preimplantation genetic testing for monogenic disease, PGT‐M) for carrier couples. The aim of PND is to provide an accurate result as early in pregnancy as possible, which necessitates prior identification of the parental disease‐causing mutations, as well as safe and timely biopsy of fetal material. PGT‐M aims to characterize the genetic status of in vitro fertilized embryos during assisted reproductive technology (ART), in a few cells biopsied from oocytes/zygotes or embryos, in order to initiate an unaffected pregnancy. Another application of PGT‐M is preimplantation genetic diagnosis for human leukocyte antigen (PGD‐HLA), which, in addition to identifying unaffected embryos, also characterizes the embryos that are HLA compatible with an existing affected child requiring a hemopoietic stem cell transplantation (HSCT). This review outlines the current practices related to these procedures, with emphasis on the aspects related to laboratory techniques. Finally, future prospects related to developments in noninvasive prenatal diagnosis are discussed. [ABSTRACT FROM AUTHOR]

The current influx of economic migrants and asylum seekers from countries with a high prevalence of haemoglobinopathies creates new challenges for health care systems and diagnostic laboratories. The migration of carriers introduces new and novel haemoglobinopathy mutations to the diagnostic repertoire of a laboratory, often creating new pressures to improve and update the carrier screening technology and diagnostic scope. For antenatal screening programmes, the marriage of partners from different ethnic groups can lead to the risk of compound heterozygote children being born novel mutation combinations, creating problems in the provision of accurate advice regarding the expected phenotype of the thalassaemia or haemoglobinopathy disorder. In the UK, the impact of immigration required the National Haemoglobinopathy Reference laboratory to change the strategy and techniques used for the molecular diagnosis of thalassaemia and the haemoglobinopathies. In 2005, due to the increasingly large range of β-thalassaemia mutations that needed to be diagnosed, the laboratory switched from a three-step screening procedure using ARMS-PCR to a simpler but more expensive one-step strategy of DNA sequencing of the beta and alpha globin genes for all referrals. After ten years of employing this strategy, a further 57 novel thalassaemia and haemoglobionpopthy alleles were discovered (11 new β-chain variants, 15 α-chain variants, 19 β-thalassaemia mutations and 12 α+-thalassaemia mutations), increasing further the extremely heterogeneous spectrum of globin gene mutations in the UK population. [ABSTRACT FROM AUTHOR]

Introduction Differentiation of homozygous hemoglobin (Hb) E with and without α0-thalassemia is subtle on routine hematological ground. We examined in a large cohort of homozygous Hb E if the level of Hb A2 is helpful. Methods A total of 592 subjects with homozygous Hb E were recruited from ongoing thalassemia screening program. Additionally, five couples at risk of having fetuses with Hb Bart's hydrops fetalis who were homozygous Hb E were also investigated. Hb analysis was performed using capillary electrophoresis system. Globin genotypes were defined by DNA analysis. Results Subjects were classified into four groups including pure homozygous Hb E (n=532), homozygous Hb E/α0-thalassemia (n=48), Hb Constant Spring EE Bart's disease (n=8), and Hb EE Bart's disease (n=4). The levels of Hb A2 were found, respectively, to be 4.97±0.69, 6.64±1.02, 4.86±0.87, and 7.60±1.04%. Among five couples at risk, α0-thalassemia was identified in three subjects with Hb A2>6.0%. Conclusions Increased Hb A2 level is a useful marker for differentiation of homozygous Hb E with and without α0-thalassemia. This should lead to a significant reduction in number of referral cases of homozygous Hb E for molecular testing of α0-thalassemia in routine practice. [ABSTRACT FROM AUTHOR]

Since 1993, following the National Thalassemia Major Prevention Program and an increase in immigration and interracial marriages, especially in southern Taiwan, the distribution of hemoglobinopathies may have changed. This study investigates the epidemiologic transition of hemoglobinopathies. We analyzed 1870 specimens collected between 2003 and 2012 in southern Taiwan, used gap-polymerase chain reaction and PCR-restriction fragment length polymorphism-based methods, and confirmed genotypes of hemoglobinopathies by DNA sequencing. We found a 91% reduction in the incidence of thalassemia major compared with samples from between 1986 and 1995. The most common genotypes of α-thalassemia and α Hb variants were the SEA type (69.4%) and Hb Quong Sze (1.54%). The most common genotypes of β-thalassemia and β Hb variants were IVS-II-654 (46.2%) and Hb E (2.2%), respectively. Compared with studies performed in different areas of and time intervals in Taiwan, a higher prevalence of -α3.7, Hb Quong Sze, and Hb E and a lower prevalence of the SEA type were found in this study. However, the SEA type remained the most common genotype observed. In addition, an increasing number of cases with an -α3.7 type carrier, Hb Quong Sze carrier, and Gγ(Aγδβ)° were identified following a peak of interracial marriages between 2003 and 2005, reflecting a regional difference and the impact of interracial marriage. In conclusion, global migration and international marriage have changed the distribution of hemoglobinopathies in Taiwan. A more comprehensive prenatal screening for new immigrants with a longer follow-up is warranted. [ABSTRACT FROM AUTHOR]

Although commonly assessed in the context of microcytosis or sickling syndrome screening, hemoglobin mutations may not be as readily considered as a cause of other symptoms. These include macrocytosis with or without anemia, chronic or episodic hemolysis, neonatal anemia, erythrocytosis, cyanosis/hypoxia and methemoglobinemia/sulfhemoglobinemia. Hemoglobin disorders commonly interfere with the reliability of Hb A1c measurement. Because the clinical presentation can be varied and the differential diagnosis broad, a systematic evaluation guided by signs and symptoms can be effective. A tertiary care reference laboratory is particularly challenged by the absence of pertinent clinical history and relevant laboratory findings, and appropriate use of resources in a data vacuum can be problematic. To address these issues, our laboratory has constructed testing panels with a tiered strategy utilizing screening assays that detect the most common causes and reflexing additional assays that assess less common etiologies. See Figure 1. Our testing algorithm panels include a rapid hemoglobin fraction monitoring test, a generic diagnostic hemoglobin electrophoresis profile, and more specific diagnostic evaluations for microcytic anemia, hereditary hemolytic anemia, methemoglobinemia and sufhemoglobinemia and erythrocytosis. Use of these testing strategies has facilitated the identification of rare and complex hemoglobin disorders from a wide variety of ethnic groups, including over 500 distinct named alpha, beta and gamma variants (of which 60+ were novel variants at the time of first detection), 99 beta thalassemia mutations and greater than 20 large deletional beta globin cluster deletion subtypes. [ABSTRACT FROM AUTHOR]

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Objective: England has universal screening for sickle cell disease in newborns, with a policy of reporting beta thalassaemia major where it is detected. We report a case with a high gamma/beta ratio (20.167), but all other tandem mass spectrometry results within expected normal limits. The second test did not support the presumed diagnosis of beta thalassaemia major. As the two results were discrepant, further investigations were carried out. Methods and results: Sanger sequencing identified a novel mutation HBB:c.40 G > A (Hb Tower Hamlets) with concomitant beta plus (severe) thalassaemia due to the HBB:c.92 + 5 G > C mutation. Conclusions: This case highlights considerations for policy makers and implementers of new methods in screening programmes. For the English screening programme, the importance of adherence to the two-part testing strategy for newborn haemoglobinopathy screening is demonstrated. When discrepancies occur in results between the two tests, a thorough investigation is indicated. Communication, training, incident review and oversight are vital aspects of any screening programme. Protocols, risk assessments and guidance require constant monitoring and review to ensure that they remain fit for purpose. When new methods are introduced, stringent application of these principles is recommended. [ABSTRACT FROM AUTHOR]

Background A novel double heterozygosity for the α chain variant Hb Q India and β chain variant Hb S is described. Hb S is prevalent in the central part of India while Hb Q India in its heterozygous state is found mainly in Sindhi families. Methods Identification of both the variants, Hb S and Hb Q India, was done based on chromatograms of HPLC and capillary zone electrophoresis (CE). Confirmation of variants was done by PCR based amplification refractory mutation system (ARMS) technique. Results Both HPLC and CE confirmed the presence of Hb S. HPLC showed a pointed narrow peak of Hb Q India at retention time of 4.55 min while it is eluted in Hb D zone on CE. A hybrid variant of these α and β globin chains was eluted in Hb C window and Hb C zone on HPLC and CE respectively. Molecular studies using ARMS technique confirmed these findings. Both the cases showed positive sickling test and presented with mild anemia. Conclusion This is a unique 2 index cases for compound heterozygosity of Hb S with Hb Q India. [ABSTRACT FROM AUTHOR]

The molecular defects underlying haemoglobinopathies are both deletions and point mutations in the alpha- or beta-globin genes or gene-clusters. To detect point mutations causing alpha- or beta-thalassaemia, direct sequencing is the method of choice to detect the widest spectrum of molecular defects. The most established approach in DNA diagnostics to screen for the most common deletion defects causing alpha-thalassaemia or beta-thalassaemia is gap-PCR, Multiplex Ligation-dependent Probe Amplification (MLPA) and Sanger Sequencing technology to detect breakpoint sequences of previously uncharacterized deletions/duplications. We demonstrate the recent advances in the determination of duplications and deletions causing alpha- or beta-thalassemia, using Next Generation Sequencing, array Comparative Genome Hybridization and Target Locus Amplification. We present three cases in which the use of advanced technologies allow the diagnosis of unexpected disease genotypes. [ABSTRACT FROM AUTHOR]