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Test ID: THEVP    
Thalassemia and Hemoglobinopathy Evaluation

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Method Description Describes how the test is performed and provides a method-specific reference

Hemoglobin A2 and F, Blood:

Hemolysate of whole blood is injected into an analysis stream passing through a cartridge containing diethylaminoethyl-resin using HPLC. A pre-programmed 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)

 

Hemoglobin Electrophoresis, Blood:

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 electrophoregrams 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)

 

Ferritin, Serum:

The instrument used is a Beckman Coulter Unicel DXI 800. The Access Ferritin assay is a 2-site immunoenzymatic ("sandwich") assay. A sample is added to a reaction vessel with goat antiferritin-alkaline phosphatase conjugate, and paramagnetic particles coated with goat antimouse:mouse antiferritin complexes. Serum ferritin binds to the immobilized monoclonal antiferritin on the solid phase, while the goat antiferritin enzyme conjugate reacts with different antigenic sites on the ferritin molecules. After incubation in a reaction vessel, materials bound to the solid phase are held in a magnetic field, while unbound materials are washed away. Chemiluminescent substrate Lumi-Phos 530 is added to the vessel and light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of ferritin in the sample. The amount of analyte in the sample is determined from a stored, multipoint calibration curve.(Package insert: Beckman Coulter Inc, Fullerton, CA 2009)

 

Hemoglobin Electrophoresis, Molecular:

Multiplex ligation-dependent probe amplification is utilized to test for the presence of large deletions in the beta-globin gene.(Unpublished Mayo method)

 

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)

 

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)

 

Hemoglobin Variant by Mass Spectrometry:

Mass spectrometry (MS) is performed using a quadrupole-time-of-flight MS (Q-ToF Premie Waters Corp, Milford, Mass, USA) and results are analyzed with Waters BioPharmalynx software. Whole blood is diluted 1:50 with purified water and cell debris removed by centrifugation. The supernatant is then diluted 1:10 with running buffer (1:1 water: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 been integrated into a database containing historic data of multiple method measurements and empiric MS mass peaks were used as a search criterion.(Zanella-Cleon I, Joly P, Becchi M, Francina A: Phenotype determination of hemoglobinopathies by mass spectrometry. Clin Biochem 2009;42(18):1807-1817)

PDF Report Indicates whether the report includes an additional document with charts, images or other enriched information

No

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 Friday; Varies

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.

2-25 days (if Structural or Molecular are required)

Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result

25 days

Performing Laboratory Location The location of the laboratory that performs the test

Rochester