Beta thalassemia syndromes are a group of hereditary blood disorders. They are characterized by reduced or absence of globin chain in Hb, which results in reduced Hb in RBC. Mode of inheritance is autosomal recessive. Beta-Thalassemia with associated Hb anomalies HbC/Beta Thalassemia, HbE/Beta Thalassemia, HbS/Beta Thalassemia, Hereditary persistent fetal Hb(HPFH) and beta-thalassemia. Beta thalassemia trait patients are asymptomatic and acts as carriers. They may present with mild anemia. In rare case of Beta thalassemia trait defect does not lie only in beta globin chain. When associated with other syndromes a molecular defect is identified in the gene encoding transcription factor TFIIH (Beta thalassemia trait associated with tricothiodystrophy), or X-linked factor GATA-1(Thrombocytopenia with Thalassemia). The gene for beta globin chain is located in chromosome 11 .The point mutation of the gene results in reduced amount (β+), or absence of β-chain(β0) , leads to relative excess of unbound alpha globulin chain and cause beta-Thalassemia These mutations may be homozygous or heterozygous. In both these conditions there is relative excess of α chains.. Excess chains damage the red cell precursors leading to anemia.
The evaluation of the thalassemia test begins with clinical history, hematologic evaluation - Hemoglobin (gm%), PCV(Hct)in % and RBC indices which includes MCV, MCH , MCHC and RDW. The thalassemia profile test includes the hemoglobin electrophoresis at alkaline pH and quantification of HbA2 and HbF, plays an important role for diagnosis of beta-thalassemia trait. Though electrophoresis at alkaline and acid pH was done for many years, cation-exchange HPLC is emerging as the method of choice for quantification of HbA2 and HbF.
Laboratory investigations for thalassemia tests are performed on the anti- coagulated venous blood samples with EDTA. Samples should be stored at 4° C and should be tested within a week, as prolonged storage leads denaturation of Hemoglobin . Red cell indices are critical to diagnose thalassemias. The main key components of the thalassemia blood tests are Hb, RBC numbers, mean corpuscular volume (MCV) and red cell distribution width (RDW). Thalassemias are microcytic hypochromic. Key diagnostic indicator for the thalassemia test includes MCV. In adult MCV range from (80-100fl). RDW is a measure of the degree of variation in red cell size. IDA, is characterized by increase in RDW. In contrast, thalassemia trait produces the uniform microcytic pictures, without concomitant increase in RDW. In HbH disease and delta/BTT, RDW is notably increased. RDW acts as adjunct to diagnosis but not as a lone indicator. MCV<76fl, MCH<26pg and TRBC>5X106 /µL are observed as effective screening tests for beta-thalassemia trait.
Capillary Zone electrophoresis is used to determine the various abnormal hemoglobins patterns and is considered as the thalassemia diagnostic test. The principle of Electrophoresis is electrically charged molecules under an applied electric field. It plays an important role in detecting abnormal Hemoglobin. The principle is, when the proteins on the membrane are exposed to a charge gradient, hey get separated from each other and visualized in the presence of haem stain. Hb electrophoresis can be done on filter paper, cellulose acetate membrane, starch gel, citrate agar gel. They are performed on lysed packed RBC’s as no bands are formed by the presence of plasma proteins. Electrophoresis, is slow and inaccurate in the quantification of low-concentration Hb variants(HbA2 ). HbA2 levels are estimated by capillary zone electrophoresis method. The method is also termed as capillary iso-electric focusing. Here electrophoresis is carried out in a capillary tube, by permitting higher voltages .The advantage is they run on small sample size. Capillary iso-electric focusing can be automated. Has several advantages with HPLC method and gel electrophoresis method like decreased manual labour, lower cost, and can detect even minor hemoglobin component accurately. It is a high resolution with semi-automated method.It can be used for direct quantification of normal and abnormal Hb fractions.
The thalassemia profile test mainly includes the detection of HbA2 levels. HemoglobinA2, is the delta-globin chain defect and occurs in 1-2 % of the African American population. HbA2 may occur in both homozygous and heterozygous states. The main clinical significance, is failure to detect HbA2, leads to failure to diagnose β Thalassemia trait.To diagnose β Thalassemia trait both HbA and HbA2must be combined, as HbA2 is seen in small percentage, it can be missed in HPLC or gel electrophoresis alone.The main interference is HbS trait or HbSS disease, HbC trait or HbCC disease.
Interpretation of HbA2
|Sr. No.||HbA2 (%)||INTERPRETATION|
ᵟβThalassemia (HbF raised).
Rare β-Thalassemia trait.
|2.||3.4-3.7||Iron deficiency anemia with BTT.
Presence of HbS.