Hemoglobin Evaluation Reflexive Cascade

Last Literature Review: July 2020 Last Update:
  • Optimal test for the initial and confirmatory diagnosis of any suspected hemoglobinopathy in individuals who have hematologic or clinical findings suggestive of a thalassemia or hemoglobinopathy
  • Detects hemoglobin (Hb) variants
  • Not recommended for routine carrier screening in healthy adults for purposes of reproductive decision making; for population screening for hemoglobinopathies, refer to The American College of Obstetricians and Gynecologists (ACOG) practice guideline 
Reflex Pattern
  • Begins with HPLC analysis:
    • If abnormal Hb is detected, or if clinical data suggest a hemoglobinopathy, appropriate reflex testing is performed
    • A hematopathologist on the faculty of the University of Utah School of Medicine personally directs and interprets each stage of testing to completion
    • Reflex testing may include electrophoresis, solubility testing, and/or molecular analyses of globin genes

Hemoglobinopathies are a group of common, inherited disorders of hemoglobin (Hb), resulting in the synthesis of structurally abnormal globin subunits.  Some of these disorders may also cause a reduced synthesis of structurally normal globin subunits (thalassemias).  The hemoglobin evaluation reflexive cascade initially tests for abnormal hemoglobin. Additional testing, including genetic testing, is added if the results are suggestive of a hemoglobinopathy.

For typical testing strategy, refer to the Hemoglobinopathies Testing algorithm.

Disease Overview


Approximately 5% of the world’s population carries clinically important Hb variants, and 300,000 individuals with a severe hemoglobinopathy are born annually.

The most common hemoglobinopathies are beta (β) thalassemia, alpha (α) thalassemia, sickle cell Hb (HbS), HbC (common in West Africa), and HbE (common in Southeast Asia).

β thalassemia is most commonly observed in individuals from southern Europe, northern Africa, and India. Sickle cell Hb is frequently observed in Southeast Asian, Indian, and Mediterranean populations and approximately 10% of African Americans have sickle cell trait.

The carrier frequency for α thalassemia varies depending on ethnicity, as follows:

  • African, African American: 1/3
  • Middle Eastern, Southeast Asian: 1/20
  • Mediterranean: 1/30-50

Hb Barts hydrops fetalis syndrome is more frequent in Southeast Asian, Indian, and Mediterranean populations than African populations.


  • Hb is a tetrameric molecule that reversibly binds oxygen to red blood cells
  • Major adult Hb (HbA) is composed of two β-globin chains and two α-globin chains
  • Defects in the formation of the Hb complex
    • Hemoglobinopathies: structurally abnormal Hb
      • Many Hb variants have no clinical effect unless paired with a second variant
      • Reduced oxygen affinity
        • Microcytic anemia
        • Hemolytic anemia
        • Cyanosis
      • Increased oxygen affinity: erythrocytosis
    • α and β thalassemia: reduced synthesis of structurally normal globin subunits
      • Imbalance in the quantity of α and β chains


Clinical Symptoms and Laboratory Test Findings for Common Hemoglobinopathies
Hemoglobinopathy Laboratory Test Results Clinical Symptomsa
β Globin

Sickle cell anemia (HbS)

  • Homozygous for HbS

HPLC: HbS present and no HbA normocytic hemolytic anemia

Asymptomatic at birth

Episodes of vascular occlusion affecting numerous organs

Pain and swelling of hands and feet: often the first indication of the disease

Infection: frequent complication

β thalassemia minor (trait)

  • Heterozygous for β thalassemia variant

HPLC pattern in individuals ≥12 months

  • HbA is decreased: 92-95%
  • HbA2 is increased: >3.7%
  • HbF may be slightly elevated: 1-4%

MCV: reduced

Clinically asymptomatic

β thalassemia major

  • Homozygous β0 variant
  • Compound heterozygote for 2 different β0 variants

HPLC: no HbA present, HbF 95-100%

Affected individuals are transfusion dependent

Microcytic anemia, hepatosplenomegaly


  • Symptoms typically appear at 6-24 months
    • Growth retardation, failure to thrive, pallor, jaundice
  • HbF is protective in early infancy

Older individuals: leg ulcers, extramedullary hematopoiesis, thrombophilia, pulmonary arterial hypertension, endocrine dysfunction, osteoporosis

β thalassemia intermedia

  • β+ homozygote or β0/β+ compound heterozygote

HPLC pattern in individuals ≥12 months

  • HbA: 10-30%
  • HbA2: 2-5%
  • HbF: 70-90%

Milder presentation than β thalassemia major: individuals may require transfusions occasionally




Liver and spleen enlargement

Moderate/severe skeletal changes

Leg ulcers

Extramedullary masses of hyperplastic erythroid marrow

α Globin

Silent carrier

  • Loss of function of a single α-globin gene (-α/αα)

HPLC: normal

Possible mild microcytic anemia

Often clinically asymptomatic

If anemia present, may be misdiagnosed as iron deficiency

Carrier: α thalassemia trait

  • Loss of function of α-globin genes in trans (-α/-α) or in cis (--/αα)

HPLC: normal for most

Mild microcytic anemia

May have normal red cell indices

May be misdiagnosed as iron deficiency

HbH disease

  • Loss of function of 3
    α-globin genes


  • Adult: presence of HbH (β4)
  • Neonate: presence of Hb Barts (γ4)

Hemolysis with Heinz bodies

Moderate microcytic hypochromic anemia


Rare extramedullary hematopoiesis

Propensity of acute hemolysis after oxidative stress, drug therapy, or infection

Hb Barts hydrops fetalis syndrome

  • Loss of function of all 4
    α-globin genes (--/--)

HPLC: Hb Barts near 100%

Significant hemolysis

Fetal generalized edema


Pleural and pericardial effusions

Severe hypochromic anemia

Often results in fetal or perinatal death

aRelated to inadequate Hb production and accumulation of globin subunits

MCV, mean corpuscular volume



HBB (β globin), HBA1, HBA2 (α globin)


Primarily autosomal recessive, though some β-globin variants have dominant effects


  • Normal adults have two functional β-globin genes (HBB) and four functional α-globin genes (two copies each of HBA1 and HBA2)
  • 90% of α thalassemia is caused by large deletions in the HBA1 and HBA2 genes
  • -α3.7 and -α4.2 α-globin gene deletions result in deletion of a single gene
  • -(α)20.5, --SEA, --MED, --FIL, or --THAI deletions result in deletion of HBA1 and HBA2 genes from the same chromosome
  • β-globin chains with different variants may interact to alleviate or exacerbate effects of the individual variants
  • Certain deletions in the HBB gene impair the developmental switch from fetal to adult Hb, resulting in hereditary persistence of fetal Hb (HPFH)


>800 variants of Hb have been described

Test Interpretation


Varies, depending on test components


Optimal interpretation requires submission of recent CBC test results

  • Positive: one or more Hb variants detected
  • Negative: no Hb variants detected


  • Please refer to individual test components for their background and limitations.
  • May not detect all Hb variants
  • Regulatory region variants and sequence variants in genes other than HBB, HBA1, and HBA2 will not be detected
  • The phase of identified variants may not be determined
  • Specific breakpoints of large deletions/duplications will not be determined
    • May not be possible to distinguish variants of similar size
  • Individuals carrying both a deletion and a duplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies
  • Sequencing of both HBA1 and HBA2 genes may not be possible in individuals harboring large α-globin deletions on both alleles
  • Rare syndromic or acquired forms of α thalassemia associated with ATRX gene variants will not be detected
  • Diagnostic errors can occur due to rare sequence variations