Introduction

Haemoglobin types

Haemoglobin is a protein molecule made up of two pairs of two similar subunits called the globin chains, the alpha (α) globin cluster and the beta (β) globin cluster, which are located in the red blood cells (RBCs). Each red blood cell contains about 300 million haemoglobin molecules, which together weigh around 30 picograms. Haemoglobin is essential for transporting oxygen from the lungs to body tissues; its globin chains have an extremely accurate structure and are coded by genes located in the DNA on chromosomes 12 (the α gene cluster) and 11 (the β gene cluster). A partial or complete genetic defect in one or both genes may occur based on how precisely the number of α chains matches the number of β chains.¹

Thalassemia and diagnosis

The term “thalassemia” refers to a variety of inherited blood disorders (a heterogeneous group of blood diseases) that affect the haemoglobin genes. It is characterised by the absence or increase of one or more of the normal globin chains, which leads to impaired erythropoiesis (i.e. the production of RBCs). Thalassemia diagnosis includes diverse laboratory tests such as haemoglobin (Hb) analysis, DNA testing, and erythrocyte (RBCs) indices. Advanced Hb analysis provides qualitative and quantitative records by using several techniques, like capillary electrophoresis (CE) or high-performance liquid chromatography (HPLC). Unlike automated haematology analysers, which are of low accuracy in differentiating α- vs β-thalassemia, such diagnostic methods are often used for initial screening based on low Hb content and decreased erythrocyte size. The specific thalassemia mutations can be detected using reliable DNA testing methods and next-generation sequencing (NGS), producing high-specificity results.^1,2,3 

Diagnosis of thalassemias 

Clinical diagnosis

The incidence period of β-thalassemia ranges between 6 and 24 months of age, with symptoms including severely diminished erythrocyte size (microcytic anaemia), mild jaundice, and hepatosplenomegaly. However, infants develop poor growth and become pale with time. Symptoms like feeding issues, recurrent fever due to a metabolic impairment or infections, irritability, and ongoing enlargement of the abdomen due to progressive liver and spleen enlargement may occur. Thalassemia carriers are usually asymptomatic, despite developing mild anaemia sometimes. In developed countries, the clinical symptoms in late or untreated patients are pale jaundice, hepatosplenomegaly, leg ulcers, skeletal changes because of bone marrow expansion, weak muscle tone, and others.¹

Hematological diagnosis

Several laboratory tests are being used to screen or help diagnose thalassemia in heterozygous carriers. In terms of complete blood count (CBC), suspected patients demonstrate low haemoglobin (Hb) and mean corpuscular volume (MCV: the average size of RBCs) after excluding iron deficiency as the cause of anaemia.^1,3

However, peripheral blood smears further evaluate red blood cell parameters. Thalassemias can exhibit such clinical findings:

• Heinz bodies: destroyed haemoglobin inclusions
• Target cells: due to the abnormal distribution of Hb and defective RBCs’ membrane elasticity.
• Elevated percentage of reticulocytes (immature RBCs)
• Microcytic cells (MCV): small red blood cells
• Irregular RBCs due to the variation in size and shape (anisocytosis and poikilocytosis)
• Hypochromic cells: RBCs of pale colour/pigmentation. 

These parameters are more impaired in patients with β-thalassemia major compared to those with β-thalassemia intermedia. 

Iron evaluation is also conducted to rule out underlying causes of iron deficiency anaemia, such as serum iron, ferritin, and total iron binding capacity (TIBC). Erythrocyte porphyrin levels are also used to differentiate unclear beta-thalassemia minor diagnosis from iron deficiency or lead toxicity. Those with beta-thalassemia will have normal porphyrin levels, unlike those with other conditions, who will show elevated porphyrin levels.

Qualitative and quantitative haemoglobin analysis

Haemoglobin electrophoresis as capillary electrophoresis (CE), cellulose acetate electrophoresis, high-pressure liquid chromatography (HPLC), and DE-52 micro chromatography, can assess the relative amount of Hb present in red blood cells. Haemoglobin A (HbA) consists of both alpha and beta globin chains. This type of haemoglobin is responsible for up to 98% of the composition of human haemoglobin; the others, like haemoglobin A2 (HbA2) and haemoglobin F, usually make up approximately 3% of Hb and less than 2% of Hb in adults, respectively. In beta-thalassemia patients, there is a defect between the beta and alpha haemoglobin chains. Those with beta-thalassemia develop significantly higher percentages of HbF and HbA2, as well as extremely lower percentages of HbA (if not absent). Still, those with thalassemia minor usually show a mild increase of HbA2 and a slight decrease of HbA. HbA is a rare form of Hb that can develop in patients with alpha-thalassemia, but the HbS type of Hb is dominant in those with sickle cell anaemia.³

Molecular analysis

The most common mutations of the beta-globin gene are detectable by polymerase chain reaction (PCR) procedures. This method uses probes and primers with specific gene sequencing that fit the one where the genetic mutation occurs in the affected individuals; in other words, the probe complements the most common mutation in the population. The most commonly used techniques are primer-specific amplification and reverse dot plot analysis. In cases not of high-risk ancestry or if targeted analysis demonstrated there is only one variant or none, it is better to start with beta-globin gene sequencing methods, e.g., NGS or Sanger sequencing. If outcomes are unclear, follow up with deletion or duplication analysis.¹

DNA analysis

Direct DNA sequencing: The mutation can be specified via sequencing the PCR product using Sanger’s dideoxy termination method; this works through the production of the single DNA strand as a template. This can be achieved using a variety of techniques, for example, the original PCR product can be denatured and cooled immediately in order to separate the two strands of the DNA (2). 

Next-generation sequencing (NGS): NGS technologies have added significant value to the human genome characterisation. They also effectively help researchers diagnose complicated disorders through whole genome sequencing (WGS), targeted gene panels, or exome sequencing. NGS can sequence the entire human genome efficiently, which is not possible with Sanger sequencing technology. NGS involves three main steps: DNA is fragmented and linked to adaptors. Then, this fragmentation is amplified using the PCR procedure, and finally, sequencing is performed by incorporating fluorescent-labelled nucleotides using DNA  polymerases or ligation techniques. Thus, based on these NGS preparation steps, the target NGS strategy was designed to cover the entire globin gene coding regions, such as BCL11A. Initial data demonstrated that NGS could be more accurate and reliable in detecting thalassemia than ordinary diagnostic methods such as CBC, Hb analysis, and Hb typing. Additionally, a preliminary study in China showed that using PCR-NGS has revealed novel or uncommon mutations that conventional methods could not detect.²

Genetic testing of amniotic fluid is critical, mainly when both parents carry the abnormal allele. This increases the risk that their child may inherit the severe types of thalassemia, as the child will get a combination of abnormal genes. Therefore, in high-risk families, prenatal diagnosis can be conducted via two procedures: amniocentesis at 14 to 20 weeks of gestation or chorionic villi sampling at 8 to 10 weeks of gestation.³ 

Summary

• Haemoglobin is a protein molecule of two pairs of identical subunits called the globin chains, the α globin cluster and the β globin cluster, which make up the red blood cells
• Thalassemia is a heterogeneous group of blood diseases that affect the haemoglobin genes because of different mutations
• The incubation period of β-thalassemia ranges between 6 and 24 months. The clinical symptoms may include mild jaundice and hepatosplenomegaly; these symptoms can improve if thalassemia is untreated, as in developed countries
• Thalassemia carriers are asymptomatic
• Several laboratory tests, such as low haemoglobin (Hb) and mean corpuscular volume (MCV), are used to screen or help diagnose thalassemia in heterozygous carriers
• Haemoglobin electrophoresis, like capillary electrophoresis, can evaluate Hb types relative to the number of RBCs. This can help diagnose the type of thalassemia and rule out other underlying disorders
• The most relevant mutations of the beta-globin gene are detectable by polymerase chain reaction (PCR) procedures
• The target NGS strategy was designed to cover all globin gene coding regions. Preliminary data showed that NGS could be more reliable than ordinary diagnostic methods, such as CBC, in detecting thalassemia. Moreover, a preliminary study demonstrated that PCR-NGS has revealed novel or uncommon mutations that conventional approaches could not detect