Unstable Hemoglobinopathies


Indications for Testing

  • Unexplained hemolytic anemias
  • Presence of Heinz bodies detected by supravital staining of blood after more common hemoglobinopathies are ruled out by electrophoresis

Laboratory Testing

  • Initial testing – CBC with peripheral smear
  • Heat stability and/or heat denaturation – primary diagnostic test for unstable hemoglobins (isopropanol heat stability test)
    • Can be falsely positive if sample contains >5% HbF (typically neonates)
  • Heinz bodies stain – erythrocytes in peripheral blood
    • Requires incubation of erythrocytes with a supravital stain, which show variable positivity for the stain
    • Results unreliable in infants <6 months
  • Hemoglobin electrophoresis or HPLC may detect unstable hemoglobins, but many mutations are not detected
    • Unstable hemoglobin variants undergo rapid denaturation and degradation within the erythrocyte; remaining Hbs may appear relatively normal
      • Severe hemolysis with normal HPLC – consider beta globin gene sequencing
      • Rare hyperunstable hemoglobins may not be detected by any of the above tests except by globin sequencing

Differential Diagnosis

Clinical Background

Hemoglobinopathies are inherited disorders caused by mutations of the globin genes. Some mutations decrease solubility of hemoglobin and the precipitated hemoglobin decreases survival times of red blood cells (RBCs). These globin mutations are referred to as unstable hemoglobins.


  • Incidence – rare
  • Age – neonatal; usually not recognized in the first few months unless related to mutations of γ globin genes



  • Unstable hemoglobins exhibit altered solubility due to oxidation of amino acid residues in globin chains by the following pathophysiological interactions
    • Preventing formation of intact hemoglobin tetramer through weakened binding of heme to globin
    • Interfering with secondary and tertiary structures of the subunit 
    • Affecting subunit interactions (eg, interference with quaternary structure)
  • Hyperunstable hemoglobins (rare)
    • Barely detectable or undetectable in the hemolysate
    • Presumably synthesized normally but rapidly destroyed because of extreme instability, creating the phenotype of dominant inherited thalassemia
  • Heinz bodies (inclusions seen in RBCs) are the product of hemoglobin denaturation and the production of hemichromes
    • Hemichromes are generated when heme is dissociated from globin and binds elsewhere on the globin chain
    • Hemichromes attach to RBC membranes and are more likely to be destroyed in the spleen
    • End result is premature destruction of RBCs with hemolysis and possible anemia

Clinical Presentation

  • Congenital Heinz body hemolytic anemia
    • Jaundice, anemia, dark urine, leg ulcers, bilirubin gallstones
  • Neonatal syndromes [Hemoglobin Poole and hemoglobin Hasharon (γ globin mutations)]
    • Neonatal hemolysis – jaundice, anemia
    • Resolves with aging as adult hemoglobin replaces fetal hemoglobin
  • Other presentations
    • Congenital anemia, splenomegaly, pigmented gallstones
    • Mild or minimal anemia with reticulocytosis out of proportion to circulating hemoglobin
    • Drug and other oxidant-induced acute hemolysis

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Heinz Body Stain 0049090
Method: Supravital Stain

Nonspecific screen for inherited disorders

Results unreliable in infants <6 months  
Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility 0050610
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Effective test for screening and follow up of individuals  with hemoglobinopathies

Reflex pattern – if HPLC detects any abnormal peaks suggestive of a hemoglobin variant, then RBC Solubility and/or Capillary Electrophoresis will be added to aid in identification and confirmation of the variant

Sensitivity/specificity – varies, depending on test components

Quantitation of hemoglobin is recommended for a definitive diagnosis in infants 1 year and older

May not detect all hemoglobin variants

Diagnostic errors can occur due to rare sequence variations

Hemoglobin, Unstable 0049020
Method: Visual Identification

Confirm abnormal hemoglobin in presence of hemolytic anemia and suspicion of abnormal hemoglobin

Cannot be used on infants <6 months  
Beta Globin (HBB) Sequencing and Deletion/Duplication 2010117
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

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

Clinical sensitivity – 99% (~97% by sequencing and ~2% by deletion analysis) for β thalassemia and hemoglobinopathies associated with the HBB gene

Analytical sensitivity – 99%

Diagnostic errors may occur due to rare sequence variations

Breakpoints of large deletions will not be determined

Precise clinical phenotype associated with a particular deletion may not be known (eg, HPFH vs δ-β thalassemia)

Intragenic deletions in the β-globin cluster genes, other than HBB, may not be detected

Does not assess for point mutations within the coding or regulatory regions of the HBD, HBG1, HBG2, and HBE1 genes

Additional Tests Available
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Initial screen for hemoglobinopathy

Beta Globin (HBB) Gene Sequencing 0050578
Method: Polymerase Chain Reaction/Sequencing

Molecular confirmation of suspected structural hemoglobinopathy or β thalassemia

Mutations tested – complete protein coding sequence with exon/intron boundaries, proximal promoter, 5’ and 3’ untranslated regions, and intronic mutations IVS-II-654, IVS-II-705 and IVS-II-745"

Clinical sensitivity – ~97%, for β thalassemia and hemoglobinopathies associated with the HBB gene

Diagnostic errors may occur due to rare sequence variations

Beta Globin (HBB) Deletion/Duplication 2010113
Method: Multiplex Ligation-dependent Probe Amplification

Second-tier test detects large deletions of the β-globin gene cluster associated with β thalassemia or hereditary persistence of fetal hemoglobin (HPFH)

Preferred initial test is the combined sequencing and deletion/duplication test