Hemoglobinopathies

Diagnosis

Indications for Testing

• Hemolytic anemia
• Family history of hemoglobinopathy

Laboratory Testing

• Initial testing – CBC with peripheral smear demonstrating any of the following
  • Polychromasia, spherocytes, schistocytes, sickle cells, Heinz bodies, basophilic stippling; however, the lack of any of these cells does not rule out hemolytic anemia
• For thalassemia testing, see Thalassemias topic
• Many hemoglobinopathies can be diagnosed using electrophoretic or high-performance liquid chromatography (HPLC) techniques, but some are missed
  • Recommended screening tests for specific phenotypes
    • Hemoglobin instability test (isopropanol or heat instability test) for unstable hemoglobins causing hemolytic anemia
    • Determination of heme oxygen dissociation curve (P50) for those with polycythemia and decreased oxygen affinity hemoglobin
      • Testing for congenital polycythemia (VHL mutation)
    • Spectrophotometric techniques for M-hemoglobins
• P50 testing for polycythemia syndrome 
  • Some mutant hemoglobins cannot be readily identified by electrophoretic or HPLC techniques
  • Confirmation of suspected hemoglobin variants can be obtained by DNA analysis

Screening

Clinical Background

Hemoglobinopathies are a group of common, inherited disorders of hemoglobin, resulting in either the synthesis of structurally abnormal globin subunits or a reduced synthesis of structurally normal globin subunits (thalassemias).

Epidemiology

• Incidence
  • Hemoglobinopathies are among the most common monogenic diseases, with ~5% of the world’s population carrying a hemoglobin mutation
  • >300,000 children affected with severe hemoglobinopathy worldwide  

Pathophysiology

• Hemoglobin, a tetramer of two α and two β or β-like (δ and γ) globin chains found in red blood cells, combines reversibly with oxygen and is the medium by which oxygen is transported within the body
• Synthesis of globins in adults is controlled by 5 genes: α, β, γ^a, γ^g and δ
  • Primary hemoglobin (Hb) in normal adults is HbA, with small amounts of HbA₂ and HbF
  • Hemoglobinopathies are the result of mutations of the globin genes
  • Hemoglobin can also be modified by environmental factors or inherited mutations that do not affect the globin chain
    • Examples include methemoglobin, carboxyhemoglobin, and S-nitrosohemoglobin (also referred to as dyshemoglobinemia)
  • >400 hemoglobin variants have been identified as resulting from α, β, γ or δ globin gene mutations
  • Identification of the hemoglobin variant leads to correct diagnosis, improved treatment, and accurate genetic counseling
• Hemoglobin variants may produce different phenotypes
  • Benign – clinically and hematologically insignificant
  • Sickling disease – associated with several genotypes
  • Hemolytic anemias – unstable hemoglobins
  • Methemoglobinemia (M-hemoglobins) – associated with heme in oxidized ferric (Fe +3) rather than normal ferrous (Fe +2) state, which renders methemoglobin unable to deliver oxygen and results in cyanosis
    • Methemoglobin can also be acquired or inherited from cytochrome b5 reductase mutations
  • Polycythemia – associated with increased oxygen hemoglobin affinity (ie, decreased p50)
  • Anemia – associated with decreased oxygen hemoglobin affinity (ie, increased p50)
  • Thalassemia phenotype (imbalance of α and β/β-like chains, ie, HbE, Hb Constant Spring, Hb Lepore) – shares features of both hemoglobinopathies and thalassemias

Types of hemoglobinopathies

• Sickle cell disease

  Epidemiology Incidence 1/200-600 African Americans HbS causes 60-70% of sickle cell disease in U.S. (1/2,000) Ethnicity HbS occurs most commonly in sub-Saharan Africans and some ethnic and racial groups in Southern Europe (Greece, Turkey and Sicily), India, and the Middle East HbC common in West Africa HbE common in Southeast Asia Genetics Autosomal recessive Sickle cell anemia is characterized by homozygosity for HbS Sickle cell trait (Hb AS) is the asymptomatic carrier state for sickle cell disease Carrier frequency of HbS is ~10% in African Americans Sickle cell disease refers to the inheritance of HbS in combination with another β globin mutation (such as HbE, HbC, Hb Lepore, or β thalassemia) to produce a sickling phenotype Common genotypes associated with sickle cell disease HbSS – sickle cell anemia HbS/C HbS/β0 and HbS/β+ – sickle cell β thalassemia HbS/E HbS/Lepore HbS/D Punjab HbS/O Arab Parental screening for sickle cell trait and interacting hemoglobinopathies can aid in genetic counseling Clinical Presentation Asymptomatic at birth – neonate has not yet switched from fetal to adult hemoglobin (γ genes to β genes) Severity of disease varies and is influenced by genotype Acute, painful, vaso-occlusive crises (long bones, chest, and back) are common Complications – spleen fibrosis and acute splenic sequestration, cerebrovascular accident, kidney failure, lungs (acute chest syndrome and pulmonary hypertension), central nevous system and bone infarcts, tissue ischemia, priapism, gallstone disease, increased risk of invasive bacterial infections, aplastic crisis Milder forms present with hemolytic anemia

  Congenital methemoglobinemia

  Epidemiology Incidence – not common; type II is sporadic Ethnicity – type I is endemic in Asthabascan and Navajo native Americans, Yakuts, and Siberian natives; sporadic in other races Genetics Autosomal dominant disorder Mutations of α globin genes; lifelong phenotype of cyanosis   Mutations of β globin genes; phenotype of cyanosis after 2-6 months of age Mutations of γ globin genes; phenotype of cyanosis during first 2-6 months of age Autosomal recessive disorder Homozygous or compound heterozygous mutations of cytochrome b5 reductase Type I – affects mature red blood cells only Type II – affects all cell types Clinical Presentation Type I – anemia, gray skin, cyanosis; no reduced life expectancy Tolerate levels of M-hemoglobins up to 40% Type II – manifests as intellectual diversity and developmental delay; reduced life expectancy

  Polycythemia or anemia (associated with hemoglobin variants with high oxygen affinity)

  Epidemiology Incidence – rare cause of congenital polycythemia Genetics Autosomal dominant; de novo mutations have been reported More than 100 high-affinity hemoglobin variants have been described; low-affinity variants are less common Mutations resulting in hemoglobins with altered oxygen affinity may occur in either the α or β globin genes Mutations of the α globin gene resulting in hemoglobins with altered oxygen affinity have a milder clinical effect than similar mutations of the β globin gene due to the number of α gene copies Homozygotes for β globin gene mutations resulting in hemoglobins with altered oxygen affinity may be more severely affected than heterozygotes Coinheritance of a thalassemia allele may alter clinical expression Clinical Presentation High oxygen affinity variants Associated with Benign hereditary polycythemia Increased hematocrit Increased blood hemoglobin concentration Normal leukocyte and platelet counts Lack of splenomegaly Characterized by Decreased p50 value – partial pressure of oxygen where hemoglobin is 50% oxygenated Sigmoidal oxygen dissociation curve (ODC) shifted to the left Rare endemic familial congenital polycythemia variance associated with mutation of VHL gene

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

Diagnostic Algorithm

  Hemolytic Anemias Testing Algorithm