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Hemoglobin Electrophoresis 

  • Author: Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP; Chief Editor: Eric B Staros, MD  more...
 
Updated: Jan 16, 2014
 

Reference Range

Hemoglobin electrophoresis is used as a screening test to identify variant and abnormal hemoglobins, including hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin F (HbF; fetal hemoglobin), hemoglobin C (HbC), and hemoglobin S (HbS).

The reference ranges are as follows:[1]

  • HbA 1: 95%-98%
  • HbA 2: 1.5%-3.5%
  • HbF: < 2% (age-dependent)
  • HbC: Absent
  • HbS: Absent
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Interpretation

Results indicate the following:

  • Presence of HbS, but with a higher proportion of HbA than HbS: Sickle cell trait (HbAS) or sickle α-thalassemia
  • Presence of HbS and HbF, but no HbA: Sickle cell anemia (HbSS), sickle beta 0 -thalassemia (hereditary persistence of fetal hemoglobin [HPFH]), or sickle–HPFH
  • Overall higher proportion of HbS than HbA and HbF: Sickle beta + -thalassemia (most likely)
  • Presence of HbC, but with a higher proportion of HbA than HbC: HbC trait (HbAC)
  • Presence of HbC and HbF, but no HbA: HbC disease (HbCC), HbC –beta 0 -thalassemia (HbC-HPFH)
  • A higher proportion of HbC than HbA: HbC beta + -thalassemia
  • Presence of HbS and HbC: HbSC disease
  • Presence of HbH: HbH disease
  • Increased HbA 2: Beta-thalassemia minor
  • Increased HbF: Hereditary persistence of fetal hemoglobin, sickle cell anemia, beta-thalassemia, HbC disease, HbE disease [2]
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Collection and Panels

Specimen: Blood

Container: Lavender-, green-, or blue-top vacuum tube

All samples must be sent in a sealed, leak-proof container marked with a biohazard sticker to comply with Occupational Safety and Health Administration (OSHA) safety standards.

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Background

Hemoglobin electrophoresis is used as a screening test to evaluate for and identify variant and abnormal hemoglobins. Alkaline and/or citrate agar electrophoresis is the commonly used method. Separation of hemoglobins is based on variable rates of migration of charged hemoglobin molecules in an electrical field.

Hemoglobin is a tetramer with two pairs of globin chains, each containing an identical heme group. Normal adult hemoglobin (HbA) has two α- and two β-globin chains (α2 β2). Fetal hemoglobin (HbF) has two α- and two γ-globin chains (α2 γ2). Minor adult hemoglobin (HbA2) is made of two α- and two δ-globin chains (α2 δ2).

Hemoglobin electrophoresis has been superseded by more rapid, sensitive, and quantitative methods of hemoglobin separation. Cellulose acetate (CA) electrophoresis at pH 8.2-8.6 can be used to resolve common variants such as HbS and HbC, but it cannot be used to distinguish between HbS and HbDPunjab and HbGPhiladelphia.[3] Citrate agar electrophoresis at pH 6.0-6.2 provides better resolution for different hemoglobin variants.[3, 4, 5]

Murine monoclonal antibodies against human normal and variant hemoglobins may be used for identification and/or quantification of hemoglobins, such as HbF measurement with radial immunodiffusion or HbF-containing erythrocytes (F-cells) with flow cytometry.[6]

Mass spectrometry, a newer analytical technology, may be used to identify highly unstable hemoglobins that may manifest clinically as hemolytic anemia or thalassemia.[7] Small amounts of these variant hemoglobins might not be detected by diagnostic techniques used in most clinical laboratories. Additionally, mass spectrometry may provide information on posttranslational modifications, such as oxidation and glycation.[3]

Capillary electrophoresis (CE) is comparable to CA electrophoresis for reliable measurement of Hb fractions. It is suitable for screening of hemoglobinopathies in many clinical laboratories.[8]

Indications/Applications

Indications and applications of hemoglobin electrophoresis include the following:

  • Evaluation of unexplained hemolytic anemia
  • Microcytic anemia unrelated to iron deficiency, chronic disease, or lead toxicity
  • A peripheral smear with abnormal red cell features (eg, target cells or sickle cells)
  • Positive family history of hemoglobinopathy
  • Positive neonatal screen results
  • Positive results on sickle cell or solubility test

Considerations

Evaluation of a suspected hemoglobinopathy should include electrophoresis of a hemolysate to detect abnormal hemoglobins and quantification of HbA2 and HbF with column chromatography. If HbS is detected, a solubility test should be performed.

Interpretation of hemoglobin electrophoresis results should be placed in the clinical context, including the family history and results of serum iron studies, red cell morphology, hemoglobin, hematocrit, and red cell indices (eg, mean corpuscular volume). Molecular testing aids in genetic counseling of patients with thalassemia and combined hemoglobinopathies.[3]

Automated high-pressure liquid chromatography instruments are proving to be useful alternative methods for hemoglobinopathy screening.[9, 10]

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Contributor Information and Disclosures
Author

Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP Associate Professor of Medicine, St Louis University School of Medicine

Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP is a member of the following medical societies: American College of Physicians, American Medical Informatics Association, Royal College of Physicians and Surgeons of Glasgow, Royal College of Surgeons of Edinburgh, Healthcare Information and Management Systems Society

Disclosure: Nothing to disclose.

Chief Editor

Eric B Staros, MD Associate Professor of Pathology, St Louis University School of Medicine; Director of Clinical Laboratories, Director of Cytopathology, Department of Pathology, St Louis University Hospital

Eric B Staros, MD is a member of the following medical societies: American Medical Association, American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology

Disclosure: Nothing to disclose.

References
  1. Ferri FF. Ferri's clinical advisor : instant diagnosis and treatment. St. Louis: Mosby; 2012:v.

  2. Commom laboratory tests. Diagnostic Tests. Available at http://www.accessmedicine.com.ezp.slu.edu/popup.aspx?aID=3136780&searchStr=hemoglobin%20electrophoresis. Accessed: April 25, 2012.

  3. Chui DHK SM. Laboratory Diagnosis of Hemoglobinopathies and Thalassemias. In: Hoffman R BJE, Shattil SJ, Furie B, Silberstein LE, McGlave P, Heslop HE, ed. Hoffman: Hematology: Basic Principles and Practice. Edinburgh: Churchill Livingstone; 2008:

  4. Schedlbauer LM, Pass KA. Cellulose acetate/citrate agar electrophoresis of filter paper hemolysates from heel stick. Pediatrics. May 1989;83(5 Pt 2):839-842:

  5. Robinson AR, Robson M, Harrison AP, Zuelzer WW. A new technique for differentiation of hemoglobin. The Journal of laboratory and clinical medicine. Nov 1957;50(5):745-752:

  6. Dover GJ, Boyer SH. Fetal hemoglobin-containing cells have the same mean corpuscular hemoglobin as cells without fetal hemoglobin: a reciprocal relationship between gamma- and beta-globin gene expression in normal subjects and in those with high fetal hemoglobin production. Blood. Apr 1987;69(4):1109-1113:

  7. Witkowska HE, Bitsch F, Shackleton CH. Expediting rare variant hemoglobin characterization by combined HPLC/electrospray mass spectrometry. Hemoglobin. Jun 1993;17(3):227-242:

  8. Kim JE, Kim BR, Woo KS, Kim JM, Park JI, Han JY. Comparison of capillary electrophoresis with cellulose acetate electrophoresis for the screening of hemoglobinopathies. The Korean journal of laboratory medicine. Oct 2011;31(4):238-243.:

  9. Caruso D, Crestani M, Mitro N, et al. High pressure liquid chromatography and electrospray ionization mass spectrometry are advantageously integrated into a two-levels approach to detection and identification of haemoglobin variants. Clinical and laboratory haematology. Apr 2005;27(2):111-119.:

  10. Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clin Chem. Oct 2004;50(10):1736-1747:

 
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