Hemoglobin Electrophoresis
Updated: Nov 20, 2019
Author: Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP, FAMIA; Chief Editor: Eric B Staros, MD
Hemoglobin electrophoresis is used as a screening test to identify normal and abnormal hemoglobins and assess their quantity. Hemoglobin types include hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin F (HbF; fetal hemoglobin), hemoglobin C (HbC), and hemoglobin S (HbS). Hemoglobin reference ranges are as follows.
Adult/elderly: Percentage of total Hb[1] :
Children: HbF[1] :
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 beta0 -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 –beta0 -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 HbA2: Beta-thalassemia minor
Increased HbF: Hereditary persistence of fetal hemoglobin, sickle cell anemia, beta-thalassemia, HbC disease, HbE disease[2]
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.
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Hemoglobin electrophoresis has been superseded by more rapid, sensitive, and quantitative methods of hemoglobin separation.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 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
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]