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
• Hemoglobin electrophoresis or HPLC may detect unstable hemoglobins, but many mutations are not detected
• Rare hyperunstable hemoglobins may not be detected by any of the above tests except by globin sequencing

Differential Diagnosis

• Sickle cell disease
• Congenital hemolytic anemias
  • Glucose-6-phosphatase deficiency
  • Pyruvate kinase deficiency
• Iron deficiency anemia
• Thalassemia
• Autoimmune hemolytic anemia

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

• ~250 of 800 known mutations of hemoglobin are unstable
  • Hemoglobin Koln and Zurich are most prevalent
• Inherited as autosomal dominant disorders, although de novo mutations do occur

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 and pigmented gallstones
  • Mild or minimal anemia with reticulocytosis out of proportion to circulating hemoglobin
  • Drug and other oxidant-induced acute hemolysis

Tests generally appear in the order most useful for common clinical situations

General

• ACOG Practice Bulletin No. 78: hemoglobinopathies in pregnancy. Obstet Gynecol. 2007; 109 (1) :229-237.PubMed
• Betuler E. Disorders of hemoglobin structure: sickle cell anemia and related abnormalities. In Williams Hematology, 7th ed. New York: McGraw-Hill, 2006. pp. 667-700.
• Jacob HS, Winterhalter KH. 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) :2008-2016.PubMed
• Joly P, Pegourie B, Courby S, Barro C, Besson G, Cohen L, Garcia C, Francina A. Two new alpha-thalassemia point mutations that are undetectable by biochemical techniques. Hemoglobin. 2008; 32 (4) :411-417.PubMed
• Kutlar F. Diagnostic approach to hemoglobinopathies. Hemoglobin. 2007; 31 (2) :243-250.PubMed
• Murray NA, Roberts IA. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed. 2007; 92 (2) :F83-F88.PubMed
• Old JM. Screening and genetic diagnosis of haemoglobinopathies. Scand J Clin Lab Invest. 2007; 67 (1) :71-86.PubMed
• Poyart C, Wajcman H. Hemolytic anemias due to hemoglobinopathies. Mol Aspects Med. 1996; 17 (2) :129-142.PubMed
• Prchal JT, Gregg XT. Hemoglobinopathies: Methemoglobinemias, Polycythemias, and Unstable Hemoglobins. In Cecil Textbook of Medicine, 22nd ed. Philadelphia: W.B. Saunders, 2004. pp. 889-893.
• Rund D, Fucharoen S. Genetic modifiers in hemoglobinopathies. Curr Mol Med. 2008; 8 (7) :600-608.PubMed
• Sonati Mde F, Costa FF. The genetics of blood disorders: hereditary hemoglobinopathies. J Pediatr (Rio J ). 2008; 84 (4 Suppl) :S40-S51.PubMed
• Steinberg MH, Nagel RL. Unstable Hemoglobins, Hemoglobins with Altered Oxygen Affinity, Hemoglobin M, and other Variants of Clinical and Biological Interest. In In Disorders of Hemoglobin, 2nd ed. New York: Cambridge University Press, 2009. pp. 589-604.
• Wajcman H, Traeger-Synodinos J, Papassotiriou I, Giordano PC, Harteveld CL, 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) :327-349.PubMed
• Williamson D. The unstable haemoglobins. Blood Rev. 1993; 7 (3) :146-163.PubMed

Hemoglobinopathies
Hemolytic Anemias
Rh Disease
Thalassemias