Hemoglobinopathies

Indications for Testing

Laboratory evaluation for a suspected hemoglobinopathy is warranted in cases of :

• Abnormal Hb findings on newborn screening
• Abnormalities in CBCs
• A known family history of an Hb disorder

Laboratory testing for hemoglobinopathy is also used in preconception or prenatal screening and management.

Laboratory Testing

Screening

Newborn Screening

In the U.S., newborn screening involves the use of dried blood spots to assess the risk for a range of conditions requiring timely follow-up, including some Hb disorders.  For state-specific newborn screening information, refer to the Association of Public Health Laboratory’s NewSTEPs data repository. 

Newborn screening for Hb disorders typically relies on protein-based methods, although some labs may include molecular testing. Common protein-based methods used in newborn screening laboratories include gel electrophoresis, capillary electrophoresis, isoelectric focusing (IEF), and high-performance liquid chromatography (HPLC).  Importantly, newborn screening is not diagnostic, and subsequent confirmatory testing is typically required. In addition, newborn screening is not designed to detect all Hb disorders.

Carrier Screening

Carrier screening for hemoglobinopathies identifies asymptomatic individuals who carry one copy of a pathogenic Hb variant. Hemoglobinopathies are usually autosomal recessive conditions; therefore, carriers may have no symptoms, but two carrier parents have a 25% chance per pregnancy of having an affected child. Approximately one in 66 individuals in the U.S. is a carrier of an Hb disorder. 

The American College of Obstetricians and Gynecologists (ACOG) recommends offering universal hemoglobinopathy testing to all individuals planning pregnancy at their first prenatal appointment if no previous results are available; this approach replaces earlier race/ethnicity-based strategies.  The American College of Medical Genetics and Genomics (ACMG) similarly recommends an ethnicity- and population-neutral approach and includes hemoglobinopathies among conditions for which testing should be offered to all pregnant individuals or those planning pregnancy.  Testing may be performed via an Hb fractionation test (e.g., Hb electrophoresis) or through molecular genetic testing (e.g., an expanded carrier panel that includes sickle cell disease and other hemoglobinopathies). 

If both reproductive partners are carriers of the same Hb trait, referral for genetic counseling is recommended to review the risk of having a child affected by an Hb disorder and to discuss available reproductive testing options (e.g., preimplantation genetic testing, amniocentesis).

Diagnosis

Initial Evaluation

For the initial evaluation, a CBC and peripheral blood smear examination should be performed. 

A CBC is used to determine whether anemia is present, based on the Hb concentration, and assess its severity. This information helps narrow the differential diagnosis.  If microcytosis is present, as indicated by a reduced mean corpuscular volume (MCV), iron studies should be performed to help differentiate thalassemia from iron deficiency anemia; however, these conditions may coexist.  Refer to the ARUP Consult Thalassemias topic for more information on distinguishing these conditions via hematologic parameters.

The absolute reticulocyte count, used to evaluate the bone marrow erythropoietic response and help identify the presence of hemolysis, provides additional diagnostic guidance.  High reticulocyte count is seen with hemolytic anemias such as sickle cell disease or unstable Hb variants.  Importantly, carriers of certain Hb variants and thalassemias (particularly mild or silent forms) may have normal red blood cell (RBC) indices. Therefore, CBC results should always be interpreted in conjunction with other laboratory findings, clinical features, and the patient’s family history. 

Peripheral blood smear examination provides qualitative morphologic information that complements CBC findings. ^,  Certain morphologic abnormalities (e.g., sickle cells, HbC crystals, target cells, Heinz bodies) may support the diagnosis of a specific condition. 

Hemoglobin Fractionation Tests

Following the initial laboratory evaluation, Hb fractionation testing is used to quantify the proportions of physiologically normal Hb types (e.g., HbA, HbA₂, and HbF) and to detect Hb variants. The most commonly used tests include gel electrophoresis, IEF, HPLC, and capillary zone electrophoresis. 

Gel electrophoresis can identify the most common abnormal Hb types (e.g., HbS and HbC) but cannot reliably detect or distinguish all Hb variants.  IEF enables the detection and diagnosis of most hemoglobinopathies using very small blood volumes, including dried blood spots, and is therefore well suited to newborn screening programs.  HPLC is more precise than gel electrophoresis and IEF and is highly automated, allowing for high-throughput sample analysis; it is also commonly used in newborn screening programs.  Capillary zone electrophoresis integrates electrophoresis and liquid chromatography and can reliably identify the most common Hb variants. 

In β thalassemia, the initial biochemical testing often shows elevated HbA₂ (and sometimes HbF), making it a key screening marker, whereas a normal HbA₂ largely argues against typical β thalassemia (although rare variants may be missed). In contrast, α thalassemia frequently results in a normal screening test and can only be diagnosed reliably by molecular testing.

Laboratories may offer hemoglobinopathy testing that incorporates multiple methods and/or employs a reflex testing strategy to identify variants.

It is important to note that, although the above methods are typically able to detect the most common variants (e.g., HbS, HbC, HbD, and HbE), rare variants may comigrate with common variants, and therefore results should be interpreted as presumptive identifications.  Molecular genetic testing is recommended for definitive identification of Hb variants and is particularly important when the methods described above yield inconclusive results or for genetic counseling purposes. 

The accuracy of Hb fractionation testing may be affected by prematurity, previous RBC transfusions, or Hb degradation. 

Other Hemoglobin Assays

Although largely superseded by modern Hb fractionation methods, the sickle solubility test is still used in some clinical settings. This assay detects the presence of HbS and may indicate sickle cell trait or disease; however, it does not quantify or differentiate Hb fractions. As a result, it cannot distinguish between trait and disease states  and should not be used as a standalone test. It is also subject to false-negative results at low HbS concentrations (e.g., in those with severe anemia and infants younger than 6 months old). ^, 

Genetic Tests

Molecular genetic testing is used to confirm hemoglobinopathies by identifying pathogenic variants and is typically performed after initial evaluation with a CBC and Hb analysis. However, it may not be necessary when an Hb variant can be definitively characterized by Hb fractionation alone. Genetic testing is particularly valuable for resolving inconclusive or discordant results and supporting genetic counseling and risk assessment in preconception and prenatal settings.

Testing is generally performed in a stepwise manner based on suspected diagnosis. For β-globin disorders, such as sickle cell disease and β thalassemia, targeted sequencing of the HBB gene (via Sanger or next generation sequencing) is typically performed, as these conditions are most often caused by point mutations or small insertions and deletions. In contrast, α thalassemia is most commonly caused by large deletions; therefore, initial testing usually involves deletion/duplication analysis of HBA1 and HBA2, often by multiplex ligation-dependent probe amplification (MLPA). If initial testing does not identify a pathogenic variant and clinical suspicion remains high, additional or complementary testing may be pursued.

Sickle cell disease is caused by biallelic pathogenic variants in the HBB gene, most commonly p.Glu6Val.

For additional information on the genetic basis of thalassemias, refer to the ARUP Consult Thalassemias topic. For additional information on genetic testing for unstable Hb variants, refer to the ARUP Consult Unstable Hemoglobinopathies topic. Refer to the ARUP Consult Hemoglobinopathies Testing Algorithm for detailed guidance on testing strategy.

Monitoring

Children 1 year and older who are diagnosed with sickle cell disease should be assessed with a CBC with reticulocyte count; renal and liver function tests; tests for HbF concentration, baseline vitamin D status, and iron status; electrophoresis or HPLC testing for thalassemia; and RBC phenotyping in case transfusion becomes necessary. Human leukocyte antigen (HLA) genotyping should be offered to affected children and full biological siblings. 

In addition to a comprehensive clinical assessment, an annual laboratory assessment is recommended for adults diagnosed with sickle cell disease. This evaluation should include a CBC with reticulocyte count; assessment of iron status and liver function; urinalysis; and blood urea nitrogen (BUN), serum creatinine, lactate dehydrogenase (to assess for hemolysis), and vitamin D tests. Extended RBC phenotyping should be performed once if not previously completed, and ideally before transfusion becomes necessary. 

Pregnant individuals with sickle cell disease should be closely monitored by a hematologist and an obstetrician/gynecologist due to increased risk for preterm labor, thrombosis, preeclampsia, acute episodes, infection, and acute chest syndrome.