Test ID: HBELC
Hemoglobin Electrophoresis Cascade, Blood

Diagnosis of thalassemias and hemoglobin variants

Evaluation of unexplained microcytosis

Hemoglobin electrophoresis cascade will always include hemoglobin A(2) and F and hemoglobin electrophoresis.

Reflex testing-Hemoglobin electrophoresis reflex testing performed at additional charge, may include any or all of the following as indicated to identify rare hemoglobin variants present: hemoglobin S screen, unstable hemoglobin, IEF confirms, hemoglobin variant by mass spec, hemoglobin F red cell distribution, beta-globin gene, large del/dup, alpha-globin gene sequencing, and beta-globin gene sequencing.

• Thalassemia Tests
• Thalassemia/Hemoglobinopathy Information Sheet
• Informed Consent for Genetic Testing

A2F/83341: Cation Exchange/High-Performance Liquid Chromatography (HPLC)

HBEL/81428: Capillary Electrophoresis

SDEX/9180: Hemoglobin S Solubility

UNHB/9095: Isopropanol Stability

IEF/81644: Isoelectric Focusing

HPFH/8270: Flow Cytometry

MASS/60286: Mass Spectrometry (MS)

HGBMO/29374: Polymerase Chain Reaction (PCR) Analysis/Multiplex Ligation-Dependent Probe Amplification (MLPA), Polymerase Chain Reaction (PCR)/DNA Sequencing

(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: ACD (solution B), sodium heparin

Specimen Volume: 6 mL

Collection Instructions: Do not transfer blood to other containers.

Additional Information:

1. Patient's age is required.

2. Include recent transfusion information.

3. For information on thalassemias and appropriate test ordering, see Thalassemia Tests in Special Instructions.

Forms:

1. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.

2. Thalassemia/Hemoglobinopathy Information Sheet (Supply T358) in Special Instructions

3. If not ordering electronically, please submit a Hematopathology/Molecular Oncology Request Form (Supply T241) with the specimen.

A large number (>800) of variants of hemoglobin (Hb) have been recognized. They are identified by capital letters (eg, Hb A or Hb S), or by the city in which the variant was first discovered (eg, Hb Koln).

Mayo Medical Laboratories receives specimens for this test from a wide geographic area and nearly one-half of all specimens received exhibit abnormalities. The most common abnormality is an increase in Hb A2 to about 4% to 8%, which is diagnostic of beta-thalassemia minor. A wide variety of other hemoglobinopathies also have been encountered. Ranked in order of relative frequency, these are: Hb S (sickle cell disease and trait), C, E, Lepore, G-Philadelphia, H, D-Los Angeles, Koln, Constant Spring, O-Arab, and others. Hb C and S are found mostly in people from west or central Africa and Hb E and H in people from southeast Asia. Hemoglobin electrophoresis is often used in the evaluation of unexplained microcytosis, thus accounting for the frequent detection of Hb Lepore, which is relatively common in Italians and others of Mediterranean ancestry and in Hb E, which is relatively common in southeast Asians resettled in the United States; microcytosis is characteristic of both Hb Lepore and Hb E.

Alpha-thalassemia is very common in the United States, occurring in approximately 30% of African Americans and accounting for the frequent occurrence of microcytosis in persons of this ethnic group. Some alpha-thalassemias (ie, hemoglobin variants H, Barts, and Constant Spring) are usually easily identified in the hemoglobin electrophoresis protocol. However, alpha-thalassemias that are from only 1 or 2 alpha-globin gene deletions are not recognized. Unfortunately, there is no easy test for the diagnosis of these alpha-thalassemias (see AGPB/9499 Alpha-Globin Gene Analysis).

Alpha-thalassemia trait itself is a harmless condition.

HEMOGLOBIN A

1-30 days: 5.9-77.2%

1-2 months: 7.9-92.4%

3-5 months: 54.7-97.1%

6-8 months: 80.0-98.0%

9-12 months: 86.2-98.0%

13-17 months: 88.8-98.0%

18-23 months: 90.4-98.0%

> or =24 months: 95.8-98.0%

HEMOGLOBIN A2

1-30 days: 0.0-2.1%

1-2 months: 0.0-2.6%

3-5 months: 1.3-3.1%

> or =6 months: 2.0-3.3%

HEMOGLOBIN F

1-30 days: 22.8-92.0%

1-2 months: 7.6-89.8%

3-5 months: 1.6-42.2%

6-8 months: 0.0-16.7%

9-12 months: 0.0-10.5%

13-17 months: 0.0-7.9%

18-23 months: 0.0-6.3%

> or =24 months: 0.0-0.9%

VARIANT

No abnormal variants

VARIANT 2

No abnormal variants

VARIANT 3

No abnormal variants

The types of hemoglobin present are identified, quantitated, and an interpretive report is issued.

Alpha-thalassemias with only 1 or 2 alpha-globin gene deletions are not recognized by this testing protocol. AGPB/9499 Alpha-Globin Gene Analysis is required to identify 1 or 2 globin gene deletions.

Hoyer JD, Hoffman DR: The Thalassemia and hemoglobinopathy syndromes. In Clinical Laboratory Medicine. Second edition. Edited by KD McMlatchey. Philadelphia, Lippincott Williams and Wilkins, 2002, pp 866-895

HPLC:
Hemolysate of whole blood is injected into an analysis stream passing through a cartridge containing diethylaminoethyl-resin using HPLC. A preprogrammed gradient controls the elution buffer mixture that also passes through the analytical cartridge. The ionic strength of the elution buffer is raised by increasing the percentage of a second buffer. As the ionic strength of the buffer increases the more strongly retained hemoglobins elute from the cartridge. Absorbance changes are detected by a dual-wavelength filter photometer. Changes in absorbances are displayed as a chromatogram of absorbances versus time.(Huismann TH, Scroeder WA, Brodie AN, et al: Microchromotography of hemoglobins. III. A simplified procedure for the determination of hemoglobin A2. J Lab Clin Med 1975;86:700-702; Ou CN, Buffone GJ, Reimer GL, Alpert AJ: High-performance liquid chromatography of human hemoglobins on a new cation exchanger. J Chromatogr 1983;266:197-205)

Electrophoresis:
The CAPILLARYS System is an automated system that uses capillary electrophoresis to separate charged molecules by their electrophoretic mobility in an alkaline buffer. Separation occurs according to the electrolyte pH and electro-osmotic flow. A sample dilution with hemolysing solution is injected by aspiration. A high voltage protein separation occurs and direct detection of the hemoglobin protein fractions is at 415 nm which is specific to hemoglobins. The resulting electrophoregram peaks are evaluated for pattern abnormalities and are quantified as a percentage of the total hemoglobin present. Examples of position of commonly found hemoglobin fractions are, from cathode to anode: Hb A2', C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart, J, N-Baltimore and H.(Louahabi A, Philippe M, et al: Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system. Clin Chem Lab Med 2006;44[3]:340-345)

The solubility test is a screening test for sickling hemoglobins. RBCs are placed in a high-molarity phosphate buffer. Sickling hemoglobins are insoluble and give a positive test.(Fairbanks VF, Klee GG: Biochemical aspects of hematology. In Tietz Textbook of Clinical Chemistry. Third edition. Edited by CA Burtis, ER Ashwood. Philadelphia, WB Saunders Company, 1999, pp 1678-1679)

Stability-unstable hemoglobins precipitate in dilute solutions of isopropanol. Washed erythrocytes are hemolyzed and cleared by centrifugation. Isopropanol is added. The hemolysate is incubated at 37 degrees C for 20 minutes and examined for turbidity. There is no turbidity with normal hemoglobins.(Fairbanks VF, Klee GG: Biochemical aspects of hematology. In Tietz Textbook of Clinical Chemistry. Third edition. Edited by CA Burtis, ER Ashwood. Philadelphia, WB Saunders Company, 1999, pp 1685-1687)

Isoelectric focusing-hemolyzed blood is placed on a polyacrylamide gel containing ampholytes pH 6 to 8. An electrical current is applied to the gel. When hemoglobin (Hb) is in this pH gradient, it moves to its isoelectric point, the pH where its net charge is zero. Once this happens, diffusion is counteracted by the electric field and Hb variants are thus separated as bands at their different isoelectric point.(Hoyer JD, Hoffman DR: The thalassemia and hemoglobinopathy syndromes. In Clinical Laboratory Medicine. Second edition. Edited by KD McMlatchey. Philadelphia, Lippincott Williams and Wilkins, 2002, pp 866-892)

Globin-purified globin chains are dissociated into monomers by urea and then separated on the basis of charge differences by electrophoresis at both alkaline and acid pH.(Hoyer JD, Hoffman DR: The thalassemia and hemoglobinopathy syndromes. In Clinical Laboratory Medicine. Second edition. Edited by KD McMlatchey. Philadelphia, Lippincott Williams and Wilkins, 2002, pp 866-892) 

Mass spectrometry (MS):
Performed using a quadrupole-time-of-flight MS (Q-ToF Premie Waters Corp, Milford, Mass, USA). Whole blood is diluted 1 to 50 with purified water and cell debris removed by centrifugation. The supernatant is then diluted 1 to 10 with running buffer (1 to 1 water to methanol, 1% formic acid) and analyzed on a Q-TOF MS in MS mode using flow injection and a myoglobin lockmass. A calculated mass for each variant has determined.(Zanella-Cleon I, Joly P, Becchi M, Francina A: Phenotype determination of hemoglobinopathies by mass spectrometry. Clin Biochem 2009;42[18]:1807-1817) 

Hb F Distribution-a flow cytometric method with a monoclonal antibody to Hb F. Specimens are analyzed by single-color flow cytometry using florescein Anti-Hb F.(Hoyer JD, Penz CS, Fairbanks VF, Katzman JA: A flow cytometric method for measurement of Hb F in red cells: application in the evaluation of hereditary persistence of fetal hemoglobin [HPFH] and other conditions with elevated Hb F levels. Blood 1998;92:40)

If the variant cannot be identified by these techniques, other testing such as amino acid or DNA sequencing may be necessary. Beta-Globin Gene, Large Del/Dup-Multiplex ligation-dependent probe amplification is utilized to test for the presence of large deletions in the beta-globin gene.(Unpublished Mayo method) 

Alpha-globin gene sequencing-PCR amplification of the 3 exons on each of the 2 alpha globin genes on chromosome 16 is followed by direct sequence analysis of these products to detect the presence of point mutations within the alpha globin gene alleles. Results are correlated with routine studies to identify unusual alpha globin variants.(Reddy PL, Bowie LJ: Sequence-based diagnosis of hemoglobinopathies in the clinical laboratory. Clin Lab Med 1997;17[1]:85-96)

Beta-globin gene sequencing-PCR amplification of the 3 exons on each of the 2 beta globin genes on chromosome 11 is followed by direct sequence analysis of these products to detect the presence of point mutations within the beta globin gene alleles. If beta-thalassemia is detected, analysis of the intron between exons 2 and 3 will also be sequenced. Results are correlated with routine studies to identify unusual beta globin variants.(Reddy PL, Bowie LJ: Sequence-based diagnosis of hemoglobinopathies in the clinical laboratory. Clin Lab Med 1997;17[1]: 85-96)

Monday through Saturday 

Hemoglobin Electrophoresis Cascade

83020-Quantitation by electrophoresis

83021-Quantitation by HPLC

IEF Confirms

82664-Electrophoresis, not elsewhere specified (if appropriate)

Hemoglobin, Unstable, Blood

83068 (if appropriate)

Hemoglobin Variant by Mass Spectrometry (MS), Blood

83789 (if appropriate)

Hemoglobin Electrophoresis, Molecular

81257-HBA1/HBA2 (alpha globin 1 and alpha globin 2) (eg, Alpha thalassemia, Hb Bart hydrops fetalis syndrome, HBH disease) gene analysis for common deletions or variant (eg, Southeast Asian, Thai, Filipino, Mediterranean, alpha3.7, alpha4.2, alpha20.5, and Constant Spring) (if appropriate)
81401-HBB (hemoglobin, beta) (eg, sickle cell anemia, hemoglobin C, hemoglobin E), common variants (eg, HbS, HbC, HbE) (if appropriate)
81403-HBB (hemoglobin, beta, beta-globin) (eg, beta thalassemia), duplication/deletion analysis (if appropriate)
 

Hemoglobin S, Screen, Blood

85660 (if appropriate)

Hemoglobin F, Red Blood Cell Distribution, Blood

88184 (if appropriate)