Unstable Hemoglobinopathies

  • Diagnosis
  • Background
  • Lab Tests
  • References
  • Related Content

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

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

Heinz Body Stain 0049090
Method: Supravital Stain


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


May not detect all hemoglobin variants

Diagnostic errors can occur due to rare sequence variations

Hemoglobin, Unstable 0049020
Method: Visual Identification


Cannot be used on infants <6 months 

Additional Tests Available

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

General References

ACOG Committee on Obstetrics. ACOG Practice Bulletin No. 78: hemoglobinopathies in pregnancy. Obstet Gynecol. 2007; 109(1): 229-37. PubMed

Jacob H, Winterhalter K. The role of hemoglobin heme loss in Heinz body formation: studies with a partially heme-deficient hemoglobin and with genetically unstable hemoglobins. J Clin Invest. 1970; 49(11): 2008-16. PubMed

Kutlar F. Diagnostic approach to hemoglobinopathies. Hemoglobin. 2007; 31(2): 243-50. PubMed

Rund D, Fucharoen S. Genetic modifiers in hemoglobinopathies. Curr Mol Med. 2008; 8(7): 600-8. PubMed

Wajcman H, Traeger-Synodinos J, Papassotiriou I, Giordano P, Harteveld C, Baudin-Creuza V, Old J. Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia. Hemoglobin. 2008; 32(4): 327-49. PubMed

Yates A, Mortier N, Hyde K, Hankins J, Ware R. The diagnostic dilemma of congenital unstable hemoglobinopathies. Pediatr Blood Cancer. 2010; 55(7): 1393-5. PubMed

Medical Reviewers

Last Update: January 2016