At a Glance

Beta thalassemia should be suspected in a patient with a microcytic anemia with an erythrocytosis.

Beta thalassemia is a microcytic anemia that results from a mutation in one or both alleles of the beta globin gene on chromosome 11. There are approximately 180 reported mutations, which are predominantly small deletions or point mutations resulting in decreased transcription, RNA splicing defects, or translation defects.

The severity of the clinical findings in thalassemia depends on the effect of the specific mutation on production of the β globin chain. β+thalassemia denotes a mutation resulting in marked decrease in β globin chain production, whereas βo thalassemia denotes a mutation resulting in absence of β globin chain production from the affected allele. Therefore, there are 3 possible clinical disorders:

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Thalassemia minor: a heterozygous disorder in which 1 β globin allele is normal

Thalassemia major: a homozygous βoo or β++genotype

Thalassemia intermedia: a homozygous β++ genotype, where the mutation allows more β globin chain production

Patients with thalassemia minor may have only a mild microcytic, hypochromic anemia with mild jaundice and splenomegaly. The peripheral blood smear may show occasional target cells and basophilic stippling.

Patients with thalassemia major may have more significant jaundice and splenomegaly, as well as chronic bone marrow hyperplasia, which may result in skeletal changes, such as frontal bossing and a “hair on end” pattern seen on x-ray. The anemia is more severe, and the patient may be transfusion dependent. The peripheral blood smear will be markedly microcytic and hypochromic with many target cells and anisopoikilocytosis. There may be increased nucleated red blood cells and a reduced reticulocyte count.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Tests to rule out an iron deficiency anemia should be performed, as this is the most common cause of a microcytic anemia. Serum iron, total iron binding capacity (TIBC), %saturation, and ferritin should be checked.

Examination of the automated red blood cell (RBC) indices can be helpful as an initial screen to determine if the patient has iron deficiency anemia versus beta thalassemia. Patients with an erythrocytosis (RBC count >5.5 mil/μl) and microcytosis (mean cell volume (MCV) <80 fL) should be suspected of having thalassemia. If the MCV/RBC ratio is less than 13, additional testing for thalassemia should be performed. Some centers use a mean cell hemoglobin (MCH) of less than 27 pg as a cutoff to perform high pressure liquid chromatography (HPLC).

New automated hematology analyzers have a %MicroR, the percentage of RBCs with a volume less than 60 fL, and a %Hypo-He, the percentage of hypochromic RBCs with hemoglobin less than 17pg. The formula % microR-%hypoHe-RDW (M-H-R) has been shown the most sensitive method of predicting if a patient has thalassemia from the RBC indices. If M-H-R is higher than -7.6, the patient is likely to have β-thalassemia trait with a sensitivity of 100% and a specificity of 92.6%.

If the RBC indices suggest beta-thalassemia, then HPLC should be performed on lysed RBCs to measure hemoglobin A22delta2) concentration and hemoglobin F (α2gamma2) concentration. As these 2 forms of hemoglobin do not utilize beta chains, they are elevated in beta thalassemia but not in alpha thalassemia. A level of hemoglobin A2 greater than 3.5% is consistent with beta thalassemia (normal values are 1.6-3.5%). Hemoglobin F should be normal to elevated, depending on the severity of the β chain loss. Normal hemoglobin F is less than 1% outside the newborn age.(Table 1)

Table 1
Classification Hemoglobin A Hemoglobin A2 Hemoglobin F Cell Distrib of HbF
Normal Normal (97%) Normal (1.6-3.5%) Normal (<1%) Heterogeneous
HPFH Present, decreased Normal (1-2.1%) Increased (20-30%) Uniform
β+Thalassemia, heterozygous Decreased (>90%) Increased (3.5-8%) Normal or slightly increased (usually <5%) Heterogeneous
β+Thalassemia, homozygous Present, decreased Variably increased Increased (<100%) Heterogeneous
βo Thalassemia Absent Mildly increased (1.5-4%) Increased (nearly 100%) Heterogeneous
δ-βThalassemia Absent Absent Increased (100%) Heterogeneous

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Iron deficiency is the most common cause of microcytic anemia and, thus, must always be considered before making a diagnosis of beta thalassemia. Of course, the 2 may exist concurrently.

If the patient develops megaloblastic anemia or significant liver disease, the MCV and MCH may increase. In addition, the drugs zidovudine and hydroxycarbamide will cause an increase in the MCV and MCH.

Hereditary persistence of fetal hemoglobin (HPFH) will result in increased hemoglobin F, but hemoglobin A2 will be normal. An increased hemoglobin F due to beta-thalassemia can be distinguished from increased hemoglobin F due to HPFH by the distribution of hemoglobin F in red cells. In beta thalassemias, the hemoglobin F is heterogeneous in expression in erythrocytes, whereas, in HPFH, the hemoglobin F is distributed uniformly among the erythrocytes. The distribution of hemoglobin F can be measured by the Kleihauer-Betke acid elution test or by flow cytometry with a fluorescence labeled anti- hemoglobin F antibody.

What Lab Results Are Absolutely Confirmatory?

Point mutations and small deletions are most common in beta thalassemia. Therefore polymerase chain reaction (PCR)-based diagnostic methods can be used easily. The most common assay used to detect known mutations is the amplification refractory mutation system (ARMS) assay, which relies on the principle that perfectly matched primers will amplify best. To detect a specific mutation, 2 parallel reactions are run, both with a common forward primer. One reaction has reverse primer complementary to the mutant sequence, and 1 reaction has a reverse primer complementary to the wild type sequence. If the mutation is present, there will be a product in the reaction using the mutant primer. A heterozygote will have products in each reaction. Variations of this assay have been created so that multiplex reactions can be run to detect the mutations common in the patient’s population of origin.

Another version of this assay is reverse dot-blotting with allele-specific oligonuleotides(ASOs) in which wild type and mutant oligonucleotides are blotted onto a nylon membrane and labeled PCR products amplified from the patient’s genomic DNA are hybridized to the membrane.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The patient may have an as yet undescribed mutation. Denaturing gradient gel electrophoresis (DGGE) and single-stranded conformation polymorphism (SSCP) assays can be used to detect the presence of a mutation that alters the migration of the DNA. Then sequencing of the implicated region would then be performed to determine the sequence of the mutant DNA.

The beta-globin gene locus is small enough that the entire gene can be easily sequenced in 2 reactions. This is the most direct, but more time consuming, method of determining what mutation the patient has in the beta globin gene.