Hemoglobinopathies are a group of common inherited disorders of hemoglobin (Hb) which can be broadly categorized into quantitative disorders (which involve imbalance in the number of globin chains) and qualitative disorders (which result in structural Hb changes). Quantitative globin defects result in thalassemias. Qualitative defects may be benign (clinically and hematologically insignificant) or may be associated with sickle cell disease, hemoglobin instability, or changes in oxygen affinity (as in methemoglobinemia). Identification of the underlying pathogenic Hb variant(s) that leads to hemoglobinopathy is important in treatment and genetic counseling. Universal newborn screening panels include testing for sickle cell anemia, the most common hemoglobinopathy; other hemoglobinopathies may not be identified until later in life. Laboratory testing is also used in diagnosis and monitoring.
Quick Answers for Clinicians
Hemoglobinopathies are the result of mutations of the globin genes that lead to quantitative changes in hemoglobin (Hb) production (thalassemias) or structural Hb variants. These changes in turn lead to altered oxygen affinity, red blood cell (RBC) abnormalities, and hemolytic anemia. More than 1,000 Hb variants have been identified, many of which are benign (clinically and hematologically inconsequential). Sickle cell disease, the most common hemoglobinopathy, occurs when at least one HbS variant is present with a second pathogenic beta globin variant; this results in abnormal Hb. For more information on pathogenic Hb variants, see the Human Hemoglobin Variants and Thalassemias database.
Sickle cell disease presents with hemolytic anemia and intermittent occlusion of the vasculature due to broken down red blood cells (RBCs). Suggestive clinical findings include otherwise unexplained anemia, severe anemia with splenomegaly, recurrent severe pain, and stroke. Common findings on a peripheral smear include sickle cells and other abnormal RBCs. Because hemoglobin F (HbF) is present at birth, neonates with sickle cell disease are usually healthy but develop symptoms as they get older.
Multiple tests are emerging for point-of-care screening or diagnosis of sickle cell disease. These tests may be particularly useful in resource-limited settings because they are less costly and resource intensive than electrophoresis and/or high-performance liquid chromatography (HPLC). Although these point-of-care tests are promising, they are not recommended for routine use.
Indications for Testing
- Screen newborns for common hemoglobinopathies
- Perform carrier screening in individuals from high-risk populations or those with family history of disease
- Diagnose a specific hemoglobinopathy if hemolytic anemia or other suggestive clinical findings are present
- Test for sickle cell disease before surgery
Newborn screening for sickle cell disease is mandated in all U.S. states. Carrier screening is recommended for individuals belonging to high-risk populations (eg, individuals of African, Southeast Asian, and Mediterranean ancestry). Due to the high prevalence of hemoglobinopathies in certain populations and the difficulty of prenatal diagnosis, testing before conception is recommended, if possible, to optimize reproductive decision-making.
One of the following techniques may be used for initial screening: high-performance liquid chromatography (HPLC), thin-layer isoelectric focusing (IEF), or capillary electrophoresis (CE). Newborn screening is performed on dried blood spots, whereas whole blood is typically used for testing in children older than 1 year and adults. Specimens that test positive are retested via a second complementary technique (eg, with CE if the initial test used HPLC), and in infants, additional testing is performed on a second blood sample if sickle cell disease is suspected.
The first step in the evaluation of a suspected hemoglobinopathy is a CBC with peripheral smear evaluation. Polychromasia, spherocytes, schistocytes, sickle cells, Heinz bodies, or basophilic stippling may be present; however, the lack of any of these findings does not rule out hemoglobinopathies or thalassemias.
Many hemoglobinopathies can be identified using electrophoretic (eg, CE) or HPLC techniques. At least two complementary techniques must be used in identification. Hemoglobin electrophoresis should not be repeated in patients who have a previous result and who do not require therapeutic intervention or monitoring of Hb variant levels.
Although the combined sensitivity and specificity of IEF and HPLC is approximately 99%, there are limitations to these techniques (eg, results may be misleading after a blood transfusion); thus, diagnosis may require additional tests, such as molecular genetic testing. A reflexive cascade can be used to avoid a missed diagnosis.
The diagnosis of sickle cell disease is established by identification of HbS, with or without another pathogenic beta globin variant, using an Hb assay. Carriers of sickle cell disease are also most commonly identified using Hb assays (either electrophoresis or HPLC).
Molecular genetic tests can be used to confirm the diagnosis of a specific hemoglobinopathy or to definitively diagnose a hemoglobinopathy if Hb assay results are ambiguous. Hemoglobin variants are often population specific; therefore, an understanding of the patient’s family history and ethnic origin is important before performing genetic testing.
Diagnosis is confirmed by identification of known pathogenic gene variants that lead to hemoglobinopathy. The diagnosis of sickle cell disease is established by identification of biallelic pathogenic variants in the HBB gene, at least one of which is the HbS causative variant (p.Glu6Val). Prenatal diagnosis of sickle cell disease is possible if the pathogenic HBB variants in the parents are known.
Specialized Tests for Specific Hemoglobinopathies
High- or low-affinity Hbs and unstable Hbs may not be detectable using the methods described above. Instability tests can be used to evaluate for unstable Hbs that cause hemolytic anemia (see the Unstable Hemoglobinopathies topic).
|Type of Hemoglobinopathy||Specific Diseases and Hb Variants||Initial Laboratory Findings||Follow-Up Analysis Techniques|
|Sickle cell||Homozygous sickle cell disease (HbSS)
Sickle-β thalassemia (HbS/β thalassemia)
Sickle-other variant (HbS/other β-globin variant)
|Normocytic anemia, sickle cells, nucleated RBCs, target cells, Howell-Jolly bodies, other RBC abnormalities||CE, HPLC, molecular genetic testing|
|Structural hemoglobinopathy that leads to thalassemia phenotype||Hb Lepore, Hb Constant Spring, HbE||Microcytic anemia or low mean corpuscular Hb, target cells||CE, HPLC, molecular genetic testing|
|Decreased solubility (crystallization)||HbC disease (HbCC)||Irregularly contracted cells, target cells||CE, HPLC, molecular genetic testing|
|Abnormal oxygen affinity (familial erythrocytosis, familial cyanosis)||High-affinity variants (eg, Hb Chesapeake)
Low-affinity variants (eg, Hb Kansas)
|Polycythemia||Heme oxygen dissociation testing|
|Familial cyanosis/congenital methemoglobinemia||M variants||Polycythemia||Spectrophotometry|
|Unstable Hbs (congenital Heinz body hemolytic anemia)||Many (eg, Hb Koln, Hb Zurich)||See Unstable Hemoglobinopathies topic|
β thalassemia (variants lead to reduced α- or β-globin production)
|See Thalassemias topic|
α, alpha; β, beta; RBCs, red blood cells
Individuals Diagnosed with Sickle Cell Disease
Infants older than 1 year who are diagnosed with sickle cell disease should have a CBC with reticulocyte count, measurement of HbF, assessment of iron status, assessment for thalassemia via electrophoresis or HPLC, baseline vitamin D test, renal and liver function tests, and RBC phenotyping in case transfusion becomes necessary. HLA genotyping should be offered to affected children and full biological siblings.
In addition to a comprehensive clinical assessment, 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, urinalysis, and liver function, blood urea nitrogen (BUN), serum creatinine, lactate dehydrogenase (to assess for hemolysis), and vitamin D tests. Extended RBC phenotyping should be performed once in case transfusion becomes necessary.
ARUP Lab Tests
Initial test for evaluation of hemoglobinopathy
Automated Cell Count/Differential
Evaluate cellular morphology
Assess erythropoiesis in hematologic conditions
Optimal test for the initial and confirmatory diagnosis of any suspected hemoglobinopathy
Effective test for screening and follow-up of individuals with known hemoglobinopathies
High Performance Liquid Chromatography/Electrophoresis/RBC Solubility
Determine presence of HbS
High Performance Liquid Chromatography (HPLC)
Effective test for secondary confirmation of HbS
Not recommended for newborns <6 mos due to high concentration of HbF
Confirm results with Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility
Quantify HbA2 and HbF in whole blood
Aid in the management of sickle cell disease and in the identification of β thalassemia carriers
Determine percentage of HbF
Preferred test to molecularly confirm β thalassemia or a hemoglobinopathy involving the β-globin gene
Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification
Molecularly confirm suspected structural hemoglobinopathy or β thalassemia
Molecularly confirm suspected structural hemoglobinopathy or β thalassemia on fetal samples
Comprehensive test for detection of HBA1 and HBA2 variants, α thalassemia, or α thalassemia trait
Polymerase Chain Reaction/Sequencing./Multiplex Ligation-dependent Probe Amplification.
Molecularly confirm suspected Hb Lepore variant identified by hemoglobin evaluation
Carrier screen for individuals with family history of Hb Lepore
Qualitative Polymerase Chain Reaction/Qualitative Electrophoresis
Confirm cases of heterozygous or homozygous methemoglobin reductase deficiency
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