Update in Laboratory Diagnosis of Thalassemia (original) (raw)

Molecular Detection of alpha Thalassemia: A Review of Prevalent Techniques

Medeniyet Medical Journal, 2021

Alpha thalassemia (α-thalassemia) is an autosomal recessive disorder due to the reduction or absence of α globin chain production. Laboratory diagnosis of α-thalassemia requires molecular analysis for the confirmatory diagnosis. A screening test, comprising complete blood count, blood smear and hemoglobin quantification by high performance liquid chromatography and capillary electrophoresis, may not possibly detect all the thalassemia diseases. This review focused on the molecular techniques used to detect α-thalassemia, and the advantages and disadvantages of each technique were highlighted. Multiplex gap-polymerase chain reaction, single-tube multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, and loop-mediated isothermal amplification were used to detect common deletion of α-thalassemia. Furthermore, the reverse dot blot analysis and a single tube multiplex polymerase chain reaction could detect non-deletion mutation of the α-globin gene. Sanger...

Molecular Diagnostics of β-Thalassemia

Balkan journal of medical genetics : BJMG, 2012

A high-quality hemoglobinopathy diagnosis is based on the results of a number of tests including assays for molecular identification of causative mutations. We describe the current diagnostic strategy for the identification of β-thalassemias and hemoglobin (Hb) variants at the International Reference Laboratory for Haemoglobinopathies, Research Centre for Genetic Engineering and Biotechnology (RCGEB) "Georgi D. Efremov," Skopje, Republic of Macedonia. Our overall approach and most of the methods we use for detection of mutations are designed for the specific target population. We discuss new technical improvements that have allowed us to substantially reduce the average time necessary for reaching a conclusive diagnosis.

Rapid differentiation of five common α-thalassemia genotypes by polymerase chain reaction

Journal of Laboratory and Clinical Medicine, 2001

The α-thalassemias are common genetic disorders that arise from reduced synthesis of the α-globin chains. At present, large-scale carrier screening and clinically valuable antenatal detection programs have not been established for the congenital disorder α-thalassemia (α-thal). We have developed a simple nonradioactive polymerase chain reaction (PCR) approach that can detect and differentiate several common α-globin gene deletional α-thals regardless of the break points. When three primer sets were used-two gene-specific sets for the α 1 -and α 2 -globin genes and one set for the β-actin gene (serving as an internal control)-PCR products from genomic DNA were simultaneously amplified and analyzed after coamplification and gel electrophoresis. The number of α-globin genes present in the subjects was determined by the intensity of α 1 and α 2 bands normalized with that of β-actin when using densitometry. Our results demonstrate that five common genotypes of deletional α-thal are differentiated by the ratios of α 1 /β-actin and α 2 /β-actin. We also examined the feasibility of coupling this allele-specific amplification to a color-complementary assay. This easy and reproducible PCR assay is suitable for identifying α-thal carriers in screenings of large populations and improving genetic counseling. (J Lab Clin Med 2001;137:290-5) Abbreviations: α-thal = α-thalassemia; ASN = antisense [primer]; CCA = color complementary assay; GC = guanine-cytosine base pair; PCR = polymerase chain reaction; SN = sense [primer]; 3´-UTR = 3´-untranslated region; UV = ultraviolet From the Molecular and Clinical Hematology Branch, National Institute of Diabetes, Digestive and Kidney Diseases,

A stepwise α-thalassemia screening strategy in high-prevalence areas

European Journal of Haematology, 2013

Introduction: Coinheritance of a-thalassemia influences the clinical and hematological phenotypes of b-hemoglobinopathies (b-thalassemia and sickle cell disease) and when present together in significant frequency within a population, a spectrum of clinical forms is observed. Precise molecular characterization of a-thalassemia is important in understanding their disease-modifying role in b-hemoglobinopathies and for diagnostic purposes. Patients and methods: Because currently used approaches are labor/costintensive, time-consuming, error-prone in certain genotype combinations and not applicable for large epidemiological screening, we developed a systematic stepwise strategy to overcome these difficulties. We successfully applied this to characterize the a-globin gene status in 150 Omani cord blood samples with Hb Barts and 32 patients with HbH disease. Results: We observed a good correlation between a-globin genotypes and level of Hb Bart's with the Hb Bart's levels significantly higher in both deletional and non-deletional a-globin genotypes. The most common a-globin genotype in HbH cases was a TSaudi a/ a TSaudi a (n = 16; 50%) followed by Àa 3.7 /-MED (n = 10; 31%). This approach detects also the a-globin gene triplication as exemplified by the study of a family where the b-globin gene defect failed to explain the b-thalassemia intermedia phenotype. Conclusion: Molecular characterization of a-thalassemia is complex due to high sequence homology between the duplicated a-globin genes and to the existence of a variety of gene rearrangements (small and large deletions of various sizes) and punctual substitutions (nondeletional alleles). The novelty of our strategy resides, not in the individual technical steps per se but in the reasoned sequential order of their use taking into consideration the hematological phenotype as well.

Thalassemia in the laboratory: pearls, pitfalls, and promises

Clinical Chemistry and Laboratory Medicine (CCLM), 2018

Thalassemia is one of the most common hereditary disorders of the developing world, and it is associated with severe anemia and transfusion dependence. The global health burden of thalassemia has increased as a result of human mobility and migration in recent years. Depending on inherited mutations, thalassemia patients exhibit distorted hemoglobin (Hb) patterns and deviated red cell indices, both of which can be used to support identification by diagnostic tools. Diagnostic approaches vary depending on the target population and the aim of the testing. Current methods, which are based on Hb patterns, are used for first-line screening, whereas molecular testing is needed for conformation of the results and for prenatal and preimplantation genetic diagnosis. In the present paper, we review the diagnostic parameters, pitfalls, interfering factors, and methods; currently available best-practice guidelines; quality assurance and standardization of the procedures; and promising laboratory...

Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC

Clinical chemistry, 1998

The conventional approach to qualitative and quantitative analyses of hemoglobin (Hb) molecules for the diagnoses of hemoglobinopathies requires a combination of tests. We used an automated HPLC (VARIANT) system to study alpha-thalassemia and beta-thalassemia syndromes in Thailand. The beta-thalassemia short program is applicable to the diagnosis of alpha-thalassemia and beta-thalassemia disorders, including Hb H, EA Bart's disease, and EF Bart's disease, in adults, newborns, and fetuses. The system cannot quantify accurately certain Hb molecules, such as Hb H and Hb Bart's. The alpha-thalassemia short program was therefore developed and used to quantify Hb Bart's to detect alpha-thalassemia genotypes in cord blood. This automated HPLC system is an alternative approach to the diagnosis of complicated thalassemia syndromes in Thailand and Southeast Asia.

Detection of eight β-thalassemia mutations using a DNA enzyme immunoassay

International Journal of Clinical & Laboratory Research, 1996

We describe the use of a polymerase chain reaction-based method followed by a DNA enzyme immunoassay for the simultaneous detection of the eight most common ,6-thalassemia mutations in the Mediterranean population. The method is specific, sensitive, and easily applicable in routine clinical laboratories for the molecular diagnosis of,6-thalassemia patients and at risk couples.

Monitoring Molecular Heterogeneity of β-thalassemia Syndrome in District Nowshehra

Science, Technology and Development, 2016

Among the reported single gene disorders/syndrome, hemoglobin infection is the most common and widespread genetic disorder. Thalassemia arises from defect in red blood cell proteinaceous component (hemoglobin), which carries oxygen and may lead to anemia. Present study aimed to detect the common molecular abnormalities of β-thalassemia syndrome in district Nowshehra, Khyber Pakhtunkhwa, Pakistan. This work was conducted at Abdul Wali Khan University, Mardan, the reports collecting blood samples from patients (average age 13 years) and their families with β-thalassemia major (n = 13 families) belonged to district Nowshehra. The assembled blood samples from the patients were tested for the presence of most common mutations, using Polymerase Chain Reaction (PCR). This study reports six known mutations (IVS-1-5, FSC 8/9, CD 41/42, IVS-1-1, CD 15 and FSC-5) comprising 90% of the total β-thalassemia genes in Pakistan. In our study, IVS 1-5 and FSC-8/9 were the most widespread β-thalassemia gene mutation detected in patients belonging to district Nowshehra. The findings of the study can be used to get an idea about the most common mutation in this region and in designing pre-natal programmes to control the genetic disease.

Review on screening and analysis techniques for hemoglobin variants and thalassemia

Talanta, 2005

Thalassemia involves gene mutation that causes the production of an insufficient amount of normal structure globin chains while Hb variant involves gene mutation that causes the change in type or number of amino acid of the globin chain. It has been reported that some 200 million people worldwide had hemoglobinopathies of some sort. Attempts to develop effective and economical techniques for screening and analysis of thalassemia and Hb variants have become very important. In this review, we report the different techniques available, ranging from initial screening to extensive analysis, comparing advantages and disadvantages. Some indirect studies related to thalassemia indication and treatment follow-up are also included. We hope that information on these various techniques would be useful for some scientists who are working on development of a new technique or improving the existing ones.