Thalassemias

Thalassemias are inherited hemoglobinopathies that arise from the unbalanced synthesis of globin chains, which results in abnormal hemoglobin (Hb).    Thalassemias affect an estimated 5-7% of the worldwide population and are most common in individuals of Mediterranean, Middle Eastern, Southeast Asian, African, and African American descent.  The most common thalassemias are beta (β) thalassemia (caused by variants in the HBB gene that lead to decreased β globin) and alpha (α) thalassemia (caused by variants in the HBA1 and/or HBA2 genes that lead to decreased α globin).   Symptoms range from mild anemia to fatal hydrops fetalis.     Some forms of thalassemia may be associated with significant hemolysis. Laboratory testing for thalassemias includes screening and diagnosis via routine blood tests, structural Hb testing (eg, high-performance liquid chromatography [HPLC] or isoelectric focusing [IEF]), and genetic testing.

Quick Answers for Clinicians

How can iron deficiency anemia be differentiated from thalassemia?

Both iron deficiency anemia and thalassemia may present as a microcytic anemia. Iron parameters may be helpful for distinguishing between the two. For example, serum ferritin is generally low in iron deficiency anemia (in the absence of inflammation), but not in thalassemia.  See the Microcytic Anemia Testing Algorithm for additional information.

What is the recommended testing strategy for complex thalassemias?

Several complex thalassemias are known to exist, including sickle cell-beta (β) thalassemia and delta beta (δβ) thalassemia.  These conditions may produce hematologic findings or clinical symptoms, or may be asymptomatic; however, it is important to identify carriers for genetic counseling purposes.  Recommended testing for a suspected complex thalassemia is similar to that for alpha (α) and β thalassemias, and includes a CBC with peripheral smear, hemoglobin electrophoresis (or equivalent) with hemoglobin (Hb) A2 (HbA2) quantitation, HbF measurement, and HbH inclusion stain.  Genetic testing is recommended to follow up on inconclusive results, identify mild mutations for carrier screening purposes, investigate genetic interactions, and for prenatal testing. 

Why are there differences in genetic testing for alpha and beta thalassemias?

The alpha (α)-globin subunit is coded for by the HBA1 and HBA2 genes.  Normally, individuals have four functioning α-globin genes (αα/αα).  In most cases, loss of function of one of these genes is due to a deletion, although nondeletion variants do occur.  Hence, deletion/duplication analysis is the preferred test. The beta (β)-globin subunits are coded for by the HBB gene.  A lack of β globin is often caused by pathogenic single nucleotide substitutions; far less commonly, it may result from small insertions or deletions or other sequence variations in HBB,  making full gene sequencing the preferred test.

Indications for Testing

Laboratory testing for thalassemia is used to :

  • Perform carrier screening in individuals from populations with a high frequency of thalassemia or with a family history of thalassemia, or in reproductive partners of an individual diagnosed with thalassemia
  • Diagnose thalassemia in individuals with anemia or isolated microcytosis

Laboratory Testing

Screening

Carrier screening (with genetic counseling) is appropriate before pregnancy for individuals with a family history of thalassemia, in reproductive partners of known thalassemia carriers, and in individuals from populations with a high incidence of thalassemia.  Timely screening can help inform reproductive choices, ensure appropriate maternal care, and facilitate diagnosis in newborns.  Due to the wide range in disease severity, the CDC recommends screening newborns for both α and β thalassemias.  Primary screening is generally performed using HPLC or IEF, and results are confirmed using a second technique. 

Diagnosis

At least two complementary techniques (eg, a combination of HPLC and electrophoresis) should be used in the initial identification of a thalassemia.  Genetic testing may be needed for definitive diagnosis. 

Initial Evaluation

The first step in the evaluation of a suspected thalassemia is a CBC with peripheral smear.  Serum iron studies are also helpful for distinguishing between thalassemias and other microcytic anemias, particularly iron deficiency anemia.

Hematologic Parameters in Thalassemias and Iron Deficiency Anemia
Parameter Thalassemias Iron Deficiency Anemia
MCV Very low Low (70-80 fL)
RBC count Normal or high end of normal Low or low end of normal
Mentzer Index (MCV/RBC count)a <13 >13
Serum ferritinb Normal Low

aUseful in pediatric patients.

bIn the absence of inflammation.

MCV, mean corpuscular volume; RBC, red blood cell

Source: Muncie, 2009 

Hemoglobin Assays

Hb testing is appropriate in the following circumstances :

  • Hydrops fetalis
  • Anemia and low or absent HbA in a neonate or infant
  • Unexplained anemia and splenomegaly
  • Unexplained microcytosis
  • Suspected thalassemia
  • Unexplained target cells on peripheral smear

A variety of techniques can be used for Hb analysis. The two most commonly used techniques are HPLC and IEF.  Capillary zone electrophoresis, acid or alkaline gel electrophoresis, and other methods may also be used.  Each technique has advantages and disadvantages, and not all Hb variants can be detected by every technique. 

Hb electrophoresis or equivalent techniques should not be repeated in patients who have a previous test result and do not require therapeutic intervention. Repeat testing should only be used to make a more specific diagnosis,  or if results may have been complicated by the presence of donor Hb from a recent transfusion.  Severe iron deficiency anemia may reduce the HbA2 level by as much as 0.5%. 

Genetic Testing

Molecular genetic testing is recommended to confirm the results of Hb analysis.   

Alpha Thalassemia

Targeted deletion analysis for common HBA1 and HBA2 variants is recommended as a first genetic test for α thalassemia, followed by sequencing.  If neither common deletion analysis nor sequencing yields diagnostic results, uncommon deletion analysis or other genetic testing may be required. 

α Thalassemia Genotype-Phenotype Relationshipsa
Genotype Phenotype Laboratory Findings
αα/αα Normal

Normal MCV and MCH

Normal peripheral smear

Normal HbA, plus small amounts of HbA2 and HbF

α-/αα Asymptomatic carrier

Normal or slightly reduced MCV and MCH

Normal peripheral smear

0-2% Hb Barts at birth, normal HbA and HbA2

α-/α- or αα/-- Mild anemia

Reduced MCV and MCH

Normal peripheral smear

2-5% Hb Barts, normal HbA, HbA2 may be slightly reduced

α-/-- (HbH disease) Hemolysis, splenomegaly

Reduced MCV and MCH

Evidence of hemolysis on peripheral smear

2-5% Hb Barts, HbA reduced, HbA2 slightly reduced

--/-- (Hb Barts hydrops fetalis) Usually fatal

Increased MCV and MCH

85-90% Hb Barts, HbA and HbA2 absent

aOther Hb variants or duplications may influence phenotype.

MCH, mean corpuscular hemoglobin

Sources: Muncie, 2009 ; ACOG, 2007 ; Origa, 2016 

For prenatal counseling, it is important to know whether the nonfunctioning α-globin genes lie on the same chromosome (cis) or on opposite chromosomes (trans). When both parents carry a cis deletion of HBA1 and HBA2 (--/αα), the risk for lethal hydrops fetalis associated with Hb Barts in their offspring may be 1:4. 

Beta Thalassemia

More than 200 known HBB variants are categorized into two classes: β zero (β0, no β-globin chain synthesis from the affected allele) and β plus (β+, decreased β-globin chain synthesis from the affected allele).  HBB sequencing, including splice sites and other intronic regions, the proximal promoter region, and the 5’ and 3’ untranslated regions (UTRs), detects or confirms nearly all β thalassemia cases.  In rare cases, β-globin deletion/duplication analysis may be required. 

β Thalassemia Genotype-Phenotype Relationshipsa
Genotype Phenotype Laboratory Findings
β/β Asymptomatic

Normal RBC indices

Normal peripheral smear

Normal HbA levels plus small amounts of HbA2 and HbF

β/β0 or β/β+ (β thalassemia minor) Asymptomatic or mild anemia

Reduced MCV and MCH

Decreased HbA, increased HbA2

β0/β+ or β+/β+ (β thalassemia intermedia)

Clinically heterogeneous

Symptoms may include pallor, jaundice, cholelithiasis, liver and spleen enlargement, moderate/severe skeletal changes, leg ulcers, extramedullary masses of hyperplastic erythroid marrow

Iron overload may occur due to increased intestinal absorption of iron caused by ineffective erythropoiesis

Reduced MCV and MCH

Nucleated RBCs, poikilocytosis on peripheral smear

Decreased HbA, increased HbF, HbA2 may be slightly increased

β0/β0 or β+/β+ (β thalassemia major)

Symptoms begin at approximately 6 mos of age

Transfusion-dependent hemolytic anemia develops

Symptoms are similar to and more severe than β thalassemia intermedia

Iron overload due to repeated transfusion may result in serious symptoms, including organ failure

Reduced MCV and MCH

Nucleated RBCs, poikilocytosis on peripheral smear

HbA absent, dramatically increased HbF, HbA2 may be slightly increased

aOther Hb variants may influence phenotype.

Sources: Muncie, 2009 ; Origa, 2018 

Monitoring

Patients with β thalassemia major are transfusion dependent, whereas patients with HbH disease or β thalassemia intermedia may require occasional transfusion. Regular transfusion is associated with a number of complications, including infections, development of antibodies to RBCs, hemolytic reactions, and iron overload that leads to organ failure. 

Laboratory Testing to Monitor for Complications of Transfusion in Thalassemias
Complication Recommended Testing Recommended Frequency
Anemia CBC Before every transfusion
Infection Hepatitis A, B, and C, HIVa Annually
Iron overload Ferritin At least every 3 mos
Hepatic iron overload Liver iron concentration (liver biopsy, MRI) Every 6-24 mos after 1-2 yrs of transfusion, depending on patient needs
Reduced liver function Liver enzymes At least every 6 mos
Hypogonadism Serum gonadotropins; estradiol or testosterone Annually
Diabetes Fasting glucose or oral glucose tolerance test Annually
Hypothyroidism Free thyroxine, TSH Annually
Hypoparathyroidism PTH Annually
Hypercalciuria and nephrolithiasis Serum and urine calcium, vitamin D Annually

aSurveillance serologic testing is recommended; with appropriate immunization and good clinical care, infection with these diseases is rare.

MRI, magnetic resonance imaging; PTH, parathyroid hormone; TSH, thyroid-stimulating hormone

Source: Tubman, 2015 

ARUP Laboratory Tests

Initial Evaluation

First recommended test in the evaluation of suspected thalassemia

Use to evaluate cellular morphology

Use to distinguish between thalassemia and iron deficiency anemia

Hemoglobin Assays

Preferred test to evaluate and diagnose a suspected hemoglobinopathy

Begins with HPLC analysis; if an abnormal hemoglobin is detected or if the CBC data suggest hemoglobinopathy, appropriate testing (including electrophoresis, solubility testing, mutational analysis and/or sequencing) will be performed

Preferred test for screening and follow-up of a known hemoglobinopathy

Aids in the identification of β thalassemia carriers

Genetic Testing
α Thalassemia

Preferred test for confirmation of suspected α thalassemia or α thalassemia trait, including common, rare, and novel deletions or duplications of the α-globin gene cluster

Acceptable test for confirmation of suspected α thalassemia or α thalassemia trait

Comprehensive test for detection of α thalassemia or α thalassemia trait

β Thalassemia

Preferred test for molecular confirmation of β thalassemia or a hemoglobinopathy involving the β-globin gene

Use to confirm suspected structural hemoglobinopathy or β thalassemia

AlertThis is a second tier test and REQUIRES PERMISSION from ARUP's Genetic Counselor (800-242-2787, x2141) before ordering

Test for the prenatal detection of structural hemoglobinopathies and β thalassemia

Medical Experts

Contributor

Agarwal

Archana Mishra Agarwal, MD
Associate Professor of Clinical Pathology, University of Utah
Medical Director, Hematopathology and Special Genetics, ARUP Laboratories

References

Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®