Unstable Hemoglobinopathies

Diagnosis

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.

Epidemiology

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

Inheritance

Pathophysiology

  • 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
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Initial screen for hemoglobinopathy

   
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

Detect hemoglobin variants when abnormal hemoglobin suspected

Cannot be used reliably in infants <1 year

Rare mutations may not be detected

Abnormal hemoglobin variants may require additional testing, which may take up to 10 days

If normal HPLC with hemolysis or normal HPLC with high suspicion, consider beta globin gene sequencing

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) Gene Sequencing 0050578
Method: Polymerase Chain Reaction/Sequencing

Indicated in patients with severe hemolysis and normal HPLC