Hemoglobin Electrophoresis Cascade, Blood
Method Description Describes how the test is performed and provides a method-specific reference
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)
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;44:340-345)
The solubility test is a screening test for sickling hemoglobins. Red blood cells 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;42:1807-1817)
Hb F Distribution-a flow cytometric method with a monoclonal antibody to Hb F. Specimens are analyzed by single-color flow cytometry using fluorescein 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;17:85-96)
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: 85-96)
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Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Saturday
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
1 day/2 to 25 days if structural and/or molecular studies are required.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
7 days; abnormals kept for 1 month
Performing Laboratory Location The location of the laboratory that performs the test