Unstable Hemoglobinopathies

The unstable hemoglobinopathies are rare inherited hemoglobinopathies in which the solubility of hemoglobin is altered, often leading to hemoglobin precipitates (Heinz bodies), reduced red blood cell (RBC) lifespan, and hemolytic anemia. The clinical severity and presentation of the unstable hemoglobinopathies vary widely, and they are frequently underdiagnosed or misdiagnosed.  Laboratory testing for unstable hemoglobinopathies consists of an initial evaluation, specialized testing, and genetic testing.

Quick Answers for Clinicians

What is the pathophysiology of unstable hemoglobinopathy?

Most unstable hemoglobinopathies arise from point mutations in one of the globin genes and are inherited as autosomal dominant disorders, although de novo variants and other inheritance patterns have been observed.  These mutations lead to changes in hemoglobin structure that cause the hemoglobin to become unstable and precipitate within red blood cells (RBCs).  These precipitates, known as Heinz bodies, bind to the membrane of the RBC, causing premature breakdown of the affected cell within the spleen.  Hemoglobins Koln and Zurich are the most prevalent unstable hemoglobins; see the Human Hemoglobin Variants and Thalassemias database for more information on other variants.

How do unstable hemoglobinopathies present?

Unstable hemoglobinopathies may present with congenital Heinz body hemolytic anemia and pigmented urine.  Heinz bodies are not specific to unstable hemoglobinopathies and may also be found in enzymopathies and thalassemia.  Neonatal hemoglobinopathy syndromes related to pathogenic gamma globin variants (hemoglobin Poole and hemoglobin Hasharon) may present with hemolysis, jaundice, and anemia. These syndromes resolve with aging as adult hemoglobin replaces fetal hemoglobin.  Other presentations include congenital anemia, mild or minimal anemia with reticulocytosis out of proportion to the circulating hemoglobin, and acute hemolysis induced by drugs (eg, sulfonamides) or other oxidants. 

What are the hyperunstable hemoglobins, and what laboratory testing challenges do they present?

The hyperunstable hemoglobins are more unstable than typical unstable hemoglobins, and are thus very rapidly destroyed, which prevents their detection in hemolysate. Hyperunstable hemoglobinopathies present similarly to severe thalassemia, but are inherited in an autosomal dominant pattern.  The hyperunstable hemoglobins are not readily detected by the hemoglobin assays typically used to detect unstable hemoglobins, such as stability testing, isoelectric focusing (IEF), or high-performance liquid chromatography (HPLC). Because they are difficult to detect, genetic testing is often required for diagnosis.

What is glucose-6-phosphate dehydrogenase deficiency, and how is it diagnosed?

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells (RBCs) and can result in RBC hemolysis. The most frequent condition associated with G6PD deficiency is hemolytic anemia, which can be triggered by bacterial or viral infections, certain antibiotics and malaria medications, or favism (a reaction to the consumption of fava beans or inhalation of fava pollen). As in unstable hemoglobinopathies, Heinz bodies may be observed in G6PD deficiency. G6PD deficiency is diagnosed using enzymatic and molecular genetic tests. For more information, see the Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency Test Fact Sheet.

Which testing algorithms are related to this topic?

Indications for Testing

Testing for unstable hemoglobinopathies is useful in cases of unexplained hemolytic anemia or when there is a known familial pathogenic hemoglobin variant.

Laboratory Testing

Initial Evaluation

The first step in the evaluation of a suspected unstable hemoglobinopathy is a CBC with peripheral smear and reticulocyte count. The initial evaluation usually reveals normocytic anemia (ranging from mild to severe) with nonspecific findings of hemolysis.   Hemolysis may be chronic or may be induced by oxidative stress, such as exposure to sulfonamide drugs.  Brisk reticulocytosis is generally observed.  “Bite” or “blister” cells may be observed in the peripheral smear.  In severe cases, particularly when hyperunstable hemoglobins are present, the peripheral smear may reveal anisocytosis, basophilic stippling, Howell-Jolly bodies, and microspherocytes. 

If a hemolytic anemia workup is performed, characteristic findings include decreased hemoglobin, elevated unconjugated bilirubin, elevated lactate dehydrogenase, decreased haptoglobin, and disproportionately elevated aspartate aminotransferase (compared with alanine aminotransferase). 

Specialized Testing

Stability Testing

Stability testing is performed by incubating with isopropanol (isopropanol stability test) or treating with heat at 50° Celsius (heat stability test).  A precipitate will form if unstable hemoglobins are present.  A false-positive result may occur if there is much hemoglobin F present (as is typical in neonates) or in cases of methemoglobinemia. 

Heinz Bodies

Heinz bodies may be detected by supravital staining of erythrocytes in peripheral blood.  However, the absence of Heinz bodies does not rule out unstable hemoglobinopathy.  Furthermore, Heinz bodies are not specific to the unstable hemoglobinopathies, and may be observed in other conditions that lead to hemoglobin precipitates (eg, glucose-6-phosphate dehydrogenase [G6PD] deficiency).  Results of this test are unreliable in infants younger than 6 months of age. 

Hemoglobin Assays

Hemoglobin electrophoresis (eg, isoelectric focusing [IEF]) or high-performance liquid chromatography (HPLC) may reveal increased hemoglobin A2 and F.  However, unstable hemoglobin variants, particularly hyperunstable variants, undergo rapid denaturation and degradation within the erythrocyte, and remaining hemoglobins may appear relatively normal.  Therefore, a normal hemoglobin electrophoresis or HPLC result does not rule out an unstable hemoglobinopathy.   Hemoglobin electrophoresis should not be repeated in patients with a previous result who do not require intervention or monitoring. 

Genetic Testing

Globin gene sequencing is the only technique that may detect some of the rare unstable hemoglobins.  Therefore, sequencing of the globin genes, including the gamma globin gene in affected neonates, is often needed for a definitive diagnosis.  

ARUP Lab Tests

Initial Evaluation

Initial screen for hemoglobinopathy

Evaluate cellular morphology

Assess bone marrow response to anemia

Specialized Tests

Use to confirm unstable nature of hemoglobins

Use as a nonspecific screen for hemolysis due to drugs/toxins, enzyme deficiencies, thalassemias, and unstable hemoglobins

Effective test for screening and follow-up of individuals with known hemoglobinopathies

Genetic Tests

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

Confirm carrier status or diagnosis of β thalassemia or β-globinopathy in individual with clinical findings or family history of β thalassemia or hemoglobinopathy

Confirm a specific HBB variant in parents before prenatal testing

Prenatal diagnosis of β thalassemia or hemoglobinopathy

Molecular confirmation of suspected structural hemoglobinopathy or β thalassemia

Comprehensive test for detection of HBA1 and HBA2 variants, α thalassemia, or α thalassemia trait

Comprehensive test for detection of HBG1 and HBG2 variants, a rare cause of unstable hemoglobins in neonates

Medical Experts

Contributor

Agarwal

Archana Mishra Agarwal, MD
Associate Professor of Clinical Pathology, University of Utah
Medical Director, Hematopathology and Special Genetics, ARUP Laboratories

References

Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®