Hemoglobinopathies

  • Diagnosis
  • Algorithms
  • Screening
  • Background
  • Lab Tests
  • References
  • Related Topics
  • Videos

Indications for Testing

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
    • Reflexive cascade is preferred for initial screening
  • Recommended screening tests for specific phenotypes
    • For unstable hemoglobins causing hemolytic anemia – hemoglobin instability test (isopropanol or heat instability test)
    • For those with polycythemia and decreased oxygen affinity hemoglobin – determination of heme oxygen dissociation curve (P50)
      • Testing for congenital polycythemia (VHL mutation)
      • Spectrophotometric techniques for M-hemoglobins
  • Polycythemia syndrome – P50 testing
    • Some mutant hemoglobins cannot be readily identified by electrophoretic or HPLC techniques
    • Confirmation of suspected hemoglobin variants can be obtained by DNA analysis
  • Newborn screening for sickle cell disease is mandated in all states in the U.S.
  • Carrier screening is recommended for individuals belonging to high-risk ethnic groups
    • Initial screening – HPLC or thin layer isoelectric focusing (U.S. Preventive Services Task Force, 2007)

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

  • Prevalence
    • 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 alpha (α) and two beta (β) or β-like (delta [δ] and gamma [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, γa, γg 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

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.

Hemoglobin Evaluation Reflexive Cascade 2005792
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility/Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer/Sequencing

Limitations 

May not detect all Hb variants

Regulatory region variants and sequence variants in genes other than HBB, HBA1, and HBA2 will not be detected

The phase of identified variants may not be determined

Specific breakpoints of large deletions/duplications will not be determined, and it may not be possible to distinguish variants of similar size

Individuals carrying both a deletion and duplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies

Sequencing of both HBA1 and HBA2 genes may not be possible in individuals harboring large α-globin deletions on both alleles

Rare syndromic or acquired forms of α thalassemia associated with ATRX gene variants will not be detected

Diagnostic errors can occur due to rare sequence variations

Hemoglobin Evaluation with Reflex to Electrophoresis and/or RBC Solubility 0050610
Method: High Performance Liquid Chromatography/Electrophoresis/RBC Solubility

Beta Globin (HBB) HbS, HbC, and HbE Mutations 0051421
Method: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Limitations 

Diagnostic errors can occur due to rare sequence variations

Detects only the 3 most common missense variants in the β-globin gene

Other β- and α-globin variants are not identified

Beta Globin (HBB) HbS, HbC, and HbE Mutations, Fetal 0051422
Method: Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer

Limitations 

Diagnostic errors can occur due to rare sequence variations

Detects only the 3 most common missense variants in the β-globin gene

Other β- and α-globin variants are not identified

Alpha Globin (HBA1 and HBA2) Deletion/Duplication 2011622
Method: Multiplex Ligation-dependent Probe Amplification

Limitations 

Breakpoints of large deletions/duplications will not be determined; therefore, it may not be possible to distinguish variants of similar size

Assay does not assess for nondeletion variants within the coding or regulatory regions of the α-globin genes

Individuals carrying both a deletion and duplication within the α-globin gene cluster may appear to have a normal number of α-globin gene copies

Rare syndromic or acquired forms of α thalassemia associated with ATRX variants will not be detected

Diagnostic errors can occur due to rare sequence variations

Alpha Thalassemia (HBA1 and HBA2) 7 Deletions 0051495
Method: Polymerase Chain Reaction/Gel Electrophoresis

Limitations 

Rare α-globin gene deletions, nondeletional variants, gene duplications, and variants of the regulatory region will not be detected

Diagnostic errors can occur due to rare sequence variations

Beta Globin (HBB) Sequencing and Deletion/Duplication 2010117
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Limitations 

Diagnostic errors may occur due to rare sequence variations

Breakpoints of large deletions will not be determined

Precise clinical phenotype associated with a particular deletion may not be known (eg, HPFH vs δ-β thalassemia)

Intragenic deletions in the β-globin cluster genes, other than HBB, may not be detected

Does not assess for point variants within the coding or regulatory regions of the HBD, HBG1HBG2, and HBE1 genes

Oxygen Dissociation (P50) by Hemoximetry (Temporary Referral as of 08/30/16) 2002984
Method: Spectrophotometry/Clark Electrode

Limitations 

A simultaneously drawn control specimen from a healthy, nonsmoking individual who is not biologically related to the patient is required for meaningful interpretation

Specimens must be analyzed within 48 hours of collection; P50 value decreases with time

Will not distinguish between the possible causes for decreased P50 value, which include 2,3-DPG deficiency; high-oxygen-affinity Hb variants; methemoglobinemia; carboxyhemoglobinemia

Decreased P50 values in chronic smokers (carboxyhemoglobinemia) should be interpreted with caution; Hb has higher affinity for carbonmonoxy than for oxygen

P50 values should be correlated with age; HbF may produce a decreased P50 level

Hereditary Persistence of Fetal Hemoglobin (HPFH) 8 Mutations 2005408
Method: Polymerase Chain Reaction/Electrophoresis

Limitations 

Only the 8 targeted deletions associated with HPFH will be detected

Point variants or rare deletions that cause HPFH or δ/β thalassemia will not be identified

Other genetic modifiers of HbF levels will not be assessed

This test is unable to differentiate homozygosity for an HPFH deletion from compound heterozygosity for an HPFH deletion and a rare globin cluster deletion

Diagnostic errors can occur due to rare sequence variations

Hemoglobin Lepore (HBD/HBB Fusion) 3 Mutations 2004686
Method: Qualitative Polymerase Chain Reaction/Qualitative Electrophoresis

Limitations 

Diagnostic errors may occur due to rare sequence variation

Negative result does not exclude β thalassemia, as other β-globin gene variants are not identified by this assay

von Hippel-Lindau (VHL) Sequencing 2002970
Method: Polymerase Chain Reaction/Sequencing

Limitations 

Mosaicism, deep intronic variants, and regulatory region variants are not detected

Rare diagnostic errors may occur due to primer- or probe-site variants

Guidelines

Stephens AD, Angastiniotis M, Baysal E, Chan V, Davis B, Fucharoen S, Giordano PC, Hoyer JD, Mosca A, Wild B, International Council for the Standardisation of Haematology (ICSH). ICSH recommendations for the measurement of haemoglobin F. Int J Lab Hematol. 2012; 34(1): 14-20. PubMed

Stephens AD, Angastiniotis M, Baysal E, Chan V, Fucharoen S, Giordano PC, Hoyer JD, Mosca A, Wild B, International Council for the Standardisation of Haematology (ICSH). ICSH recommendations for the measurement of haemoglobin A2. Int J Lab Hematol. 2012; 34(1): 1-13. PubMed

Traeger-Synodinos J, Harteveld CL, Old JM, Petrou M, Galanello R, Giordano P, Angastioniotis M, De la Salle B, Henderson S, May A, EMQN haemoglobinopathies best practice meeting. EMQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies. Eur J Hum Genet. 2015; 23(4): 426-37. PubMed

US Preventive Services Task Force. Screening for sickle cell disease in newborns: recommendation statement. Am Fam Physician. 2008; 77(9): 1300-2. PubMed

General References

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

McCavit TL. Sickle cell disease. Pediatr Rev. 2012; 33(5): 195-204; quiz 205-6. PubMed

Mosca A, Paleari R, Leone D, Ivaldi G. The relevance of hemoglobin F measurement in the diagnosis of thalassemias and related hemoglobinopathies. Clin Biochem. 2009; 42(18): 1797-801. PubMed

Nascimento TS, Pereira RO, de Mello HL, Costa J. Methemoglobinemia: from diagnosis to treatment. Rev Bras Anestesiol. 2008; 58(6): 651-64. PubMed

Peters M, Heijboer H, Smiers F, Giordano PC. Diagnosis and management of thalassaemia. BMJ. 2012; 344: e228. PubMed

Sonati Md, Costa FF. The genetics of blood disorders: hereditary hemoglobinopathies. J Pediatr (Rio J). 2008; 84(4 Suppl): S40-51. PubMed

Troxler H, Kleinert P, Schmugge M, Speer O. Advances in hemoglobinopathy detection and identification. Adv Clin Chem. 2012; 57: 1-28. PubMed

Wajcman H, Riou J. Globin chain analysis: an important tool in phenotype study of hemoglobin disorders. Clin Biochem. 2009; 42(18): 1802-6. PubMed

Zanella-Cleon I, Joly P, Becchi M, Francina A. Phenotype determination of hemoglobinopathies by mass spectrometry. Clin Biochem. 2009; 42(18): 1807-17. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Agarwal AM, Nussenzveig R. Commentary. Clin Chem. 2012; 58(2): 335-6. PubMed

Agarwal N, Kutlar F, Mojica-Henshaw MP, Ou CN, Gaikwad A, Reading S, Bailey L, Kutlar A, Prchal JT. Missense mutation of the last nucleotide of exon 1 (G->C) of beta globin gene not only leads to undetectable mutant peptide and transcript but also interferes with the expression of wild allele. Haematologica. 2007; 92(12): 1715-6. PubMed

Greene DN, Vaughn CP, Crews BO, Agarwal AM. Advances in detection of hemoglobinopathies Clin Chim Acta. 2015; 439: 50-7. PubMed

Herrmann MG, Durtschi JD, Bromley K, Wittwer CT, Voelkerding KV. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem. 2006; 52(3): 494-503. PubMed

Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV. Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem. 2007; 53(8): 1544-8. PubMed

Lanikova L, Kucerova J, Indrak K, Divoka M, Issa J, Papayannopoulou T, Prchal JT, Divoky V. β-Thalassemia due to intronic LINE-1 insertion in the β-globin gene (HBB): molecular mechanisms underlying reduced transcript levels of the β-globin(L1) allele. Hum Mutat. 2013; 34(10): 1361-5. PubMed

Lanikova L, Lorenzo F, Yang C, Vankayalapati H, Drachtman R, Divoky V, Prchal JT. Novel homozygous VHL mutation in exon 2 is associated with congenital polycythemia but not with cancer. Blood. 2013; 121(19): 3918-24. PubMed

Little RR, Rohlfing CL, Hanson S, Connolly S, Higgins T, Weykamp CW, D'Costa M, Luzzi V, Owen WE, Roberts WL. Effects of hemoglobin (Hb) E and HbD traits on measurements of glycated Hb (HbA1c) by 23 methods. Clin Chem. 2008; 54(8): 1277-82. PubMed

Little RR, Vesper H, Rohlfing CL, Ospina M, Safar-Pour S, Roberts WL. Validation by a mass spectrometric reference method of use of boronate affinity chromatography to measure glycohemoglobin in the presence of hemoglobin S and C traits. Clin Chem. 2005; 51(1): 264-5. PubMed

Lorenzo FR, Yang C, Fui MN, Vankayalapati H, Zhuang Z, Huynh T, Grossmann M, Pacak K, Prchal JT. A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma. J Mol Med (Berl). 2013; 91(4): 507-12. PubMed

Lorenzo FR, Yang C, Lanikova L, Butros L, Zhuang Z, Prchal JT. Novel compound VHL heterozygosity (VHL T124A/L188V) associated with congenital polycythaemia. Br J Haematol. 2013; 162(6): 851-3. PubMed

Mongia SK, Little RR, Rohlfing CL, Hanson S, Roberts RF, Owen WE, D'Costa MA, Reyes CA, Luzzi VI, Roberts WL. Effects of hemoglobin C and S traits on the results of 14 commercial glycated hemoglobin assays. Am J Clin Pathol. 2008; 130(1): 136-40. PubMed

Niu X, Nouraie M, Campbell A, Rana S, Minniti CP, Sable C, Darbari D, Dham N, Reading S, Prchal JT, Kato GJ, Gladwin MT, Castro OL, Gordeuk VR. Angiogenic and inflammatory markers of cardiopulmonary changes in children and adolescents with sickle cell disease. PLoS One. 2009; 4(11): e7956. PubMed

Nussenzveig RH, Lingam B, Gaikwad A, Zhu Q, Jing N, Prchal JT. A novel mutation of the cytochrome-b5 reductase gene in an Indian patient: the molecular basis of type I methemoglobinemia. Haematologica. 2006; 91(11): 1542-5. PubMed

Nussenzveig RH, Vanhille DL, Hussey D, Reading S, Agarwal AM. Development of a rapid multiplex PCR assay for identification of the three common Hemoglobin-Lepore variants (Boston-Washington, Baltimore, and Hollandia) and identification of a new Lepore variant. Am J Hematol. 2012; 87(10): E74-5. PubMed

Pont-Kingdon G, Chou L, Damjanovich K, Sumner K, Herrmann M, Erali M, Lyon E. Multiplex genotyping by melting analysis of loci-spanning probes: beta-globin as an example. Biotechniques. 2007; 42(2): 193-7. PubMed

Prchal JT. Molecular basis of polycythemic disorders due to aberrant hypoxia sensing and its relevance to acute leukemia. Best Pract Res Clin Haematol. 2012; 25(4): 493-7. PubMed

Roberts WL, De BK, Brown D, Hanbury M, Hoyer JD, John G, Lambert TL, Lundell RB, Rohlfing C, Little RR. Effects of hemoglobin C and S traits on eight glycohemoglobin methods. Clin Chem. 2002; 48(2): 383-5. PubMed

Roberts WL, Safar-Pour S, De BK, Rohlfing CL, Weykamp CW, Little RR. Effects of hemoglobin C and S traits on glycohemoglobin measurements by eleven methods. Clin Chem. 2005; 51(4): 776-8. PubMed

Sable CA, Aliyu ZY, Dham N, Nouraie M, Sachdev V, Sidenko S, Miasnikova GY, Polyakova LA, Sergueeva AI, Okhotin DJ, Bushuev V, Remaley AT, Niu X, Castro OL, Gladwin MT, Kato GJ, Prchal JT, Gordeuk VR. Pulmonary artery pressure and iron deficiency in patients with upregulation of hypoxia sensing due to homozygous VHL(R200W) mutation (Chuvash polycythemia). Haematologica. 2012; 97(2): 193-200. PubMed

Sergueeva AI, Miasnikova GY, Polyakova LA, Nouraie M, Prchal JT, Gordeuk VR. Complications in children and adolescents with Chuvash polycythemia Blood. 2015; 125(2): 414-5. PubMed

Talpaz M, Paquette R, Afrin L, Hamburg SI, Prchal JT, Jamieson K, Terebelo HR, Ortega GL, Lyons RM, Tiu RV, Winton EF, Natrajan K, Odenike O, Claxton D, Peng W, O'Neill P, Erickson-Viitanen S, Leopold L, Sandor V, Levy RS, Kantarjian HM, Verstovsek S. Interim analysis of safety and efficacy of ruxolitinib in patients with myelofibrosis and low platelet counts. J Hematol Oncol. 2013; 6(1): 81. PubMed

Tomasic NL, Piterkova L, Huff C, Bilic E, Yoon D, Miasnikova GY, Sergueeva AI, Niu X, Nekhai S, Gordeuk V, Prchal JT. The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W). Haematologica. 2013; 98(4): 560-7. PubMed

Zangari M, Fink L, Tolomelli G, Lee JC, Stein BL, Hickman K, Swierczek S, Kelley TW, Berno T, Moliterno AR, Spivak JL, Gordeuk VR, Prchal JT. Could hypoxia increase the prevalence of thrombotic complications in polycythemia vera? Blood Coagul Fibrinolysis. 2013; 24(3): 311-6. PubMed

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Last Update: August 2016