Update in Laboratory Diagnosis of Thalassemia

Abstract

Alpha- and β-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous β-thalassemia, β-thalassemia/Hb E, and Hb Bart's hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and β-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in β-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and β-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

Keywords: DNA analysis; diagnosis; hemoglobin analysis; hemoglobinopathies; thalassemia.

FIGURE 1

Pattern of hemoglobin analysis in adult blood by the high-performance liquid chromatography (HPLC) system (A–C,G–I,M–O) compared with the capillary zone electrophoresis (CE) system (D–F,J–L,P–R).

FIGURE 2

Pattern of hemoglobin analysis in cord blood by the high-performance liquid chromatography (HPLC) system (A,B,E–F) compared with the capillary zone electrophoresis (CE) system (C,D,G–H).

FIGURE 3

Family pedigree showing the co-inheritance of α-thalassemia 1 in β-thalassemia/Hb E patient (II.1) with high hemoglobin (Hb) level compared to his younger brother who is a β-thalassemia/Hb E patient without α-thalassemia (II.2).

FIGURE 4

Result of allele-specific PCR showing PCR product of normal individual which can be seen only in the reaction employing the wild-type (N) primer set. While Hb CS heterozygote will generate a band using both wild-type (N) and mutant (M) primer set. Internal control was exhibited at 526 bp.

FIGURE 5

Result of RDB showing a result of normal individual will give positive results with each wild-type sequence but not with any mutant probe (upper panel). Heterozygotes show a reaction with a single mutation dot in addition to the normal dots (middle panel), whereas homozygotes will give a positive dot with that mutant probe but not with its corresponding normal sequence (two mutant dots will be seen if the individual carries two different mutations) together with positive spots for the remaining normal probes (lower panel).

FIGURE 6

Multiplex GAP–PCR with melting curve analysis for α-thalassemia genotyping. Panel (A) shows two peaks of α2-globin and ψζ-globin fragments from normal globin genotype. Panel (B) shows three peaks of α2-globin, –^SEA, and ψζ-globin from α-thalassemia 1 heterozygote (–^SEA type). Panel (C) shows three peaks of –^THAI, α2-globin, and ψζ-globin from α-thalassemia 1 heterozygote (–^THAI type). Panel (D) shows a single peak of -α³.⁷ from α-thalassemia 2 homozygote. Panel (E) shows two peaks of –^SEA and ψζ-globin from deletional Hb H disease. Panel (F) shows a single peak of –^SEA from Hb Bart’s hydrops fetalis (α-thalassemia 1 homozygote; –^SEA type).