Test ID: THEVP
Thalassemia and Hemoglobinopathy Evaluation

Diagnosis of thalassemia

This is a consultative evaluation in which the case will be evaluated at Mayo Medical Laboratories, the appropriate tests performed at an additional charge, and the results interpreted. For information on thalassemias and appropriate test ordering, see Thalassemia Tests in Special Instructions.

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

THEV/583: Consultative Interpretation

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

HBEL/81428: Capillary Electrophoresis

FERR/8689: Immunoenzymatic Assay

IEF/81644: Electrophoresis

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.)

Forms:

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

2. 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.

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

Patient's age and sex are required. Include recent transfusion information.

Blood and serum are required.

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 15 mL

Collection Instructions: Label specimen as whole blood.

Specimen Type: Serum

Container/Tube: Red top or serum gel

Specimen Volume: 0.6 mL

Collection Instructions: Label specimen as serum.

The thalassemias are a group of autosomal recessive disorders of hemoglobin (Hb) synthesis. Normal adult Hb consists of 2 alpha globin chains (encoded by 2 pairs of alpha globin genes, each pair located on 1 of the chromosomes 16), and 2 beta globin chains (encoded by 2 beta globin genes, each located on 1 of the chromosomes 11). Thalassemia syndromes result from an underproduction of 1 or 2 types of globin chains and are characterized by the type (alpha, beta, delta) and magnitude of underproduction (number of defective genes) and the severity of clinical symptoms (minor, major). The severity of the clinical and hematologic effects is directly related to the number of genes deleted or affected.

The most common form of thalassemia is heterozygous alpha thalassemia 2, with 1 affected alpha globin gene. In heterozygous alpha thalassemia 2, there is no clinical effect and the blood count, including the mean cell volume, is normal. Heterozygous alpha thalassemia 1 and homozygous alpha thalassemia 2 (both with 2 affected genes) have the typical thalassemic picture (eg, hypochromic microcytic anemia, pallor, fatigue, shortness of breath, jaundice, and splenomegaly).

Hemoglobin H (Hb H) disease, having a deletion of 3 alpha chains, is a moderate-to-severe hemolytic disease. The severity of Hb H disease is related to the amount of Hb H in the red cells. The morphology of the red cells is often very bizarre due to denatured Hb found within the red cells.

The deletion of all 4 alpha chains is incompatible with life. Affected fetuses are hydropic and die in utero or shortly after premature birth. The blood smears show large hypochromic red cells, nucleated red cells, target cells, and red cell fragments. Hb Barts, Hb H, and Hb Portland are present in significant quantities. It is the most common cause of hydrops fetalis in Southeast Asia and southern China.

This consultative study tests for the detection of alpha-thalassemias, beta-thalassemias, delta-beta-thalassemia, and for Hb variants that are commonly accompanied by thalassemias: Hb H, Hb Lepore, Hb Barts, unstable Hb, hemolytic anemias, Hb E, hereditary persistence of high fetal Hb (several varieties), and combinations of Hb S with alpha- or beta-thalassemia, Hb E/beta-O-thalassemia, and many other complex thalassemic disorders. Some of the alpha-thalassemias (eg, Hb H disease) can be reliably identified by Hb electrophoresis alone; some require DNA probe studies. Since iron deficiency can mimic thalassemias, ferritin levels are measured to evaluate this possibility.

Definitive results and an interpretive report will be provided.

A hematopathologist expert in these disorders evaluates the case, appropriate tests are performed, and an interpretive report is issued.

DNA probe studies reveal deletional mutations that include most, but not all, alpha-thalassemias.

Hoyer JD, Hoffman DR: The thalassemia and hemoglobinopathy syndromes. In Clinical Laboratory Medicine. Second edition. Edited by KD McMlatchey. Philadelphia, Lippencott Williams and Wilkins, 2002, pp 866-892

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)

Monday through Friday; Varies

Thalassemia and Hemoglobinopathy Evaluation

82728-Ferritin

83020-Hemoglobin electrophoresis

83021-Hemoglobin A2 and F

IEF Confirms

82664 (if appropriate)

Hemoglobin, Unstable, Blood

83068 (if appropriate)

Hemoglobin Variant by Mass Spectrometry

83789 (if appropriate)

Hemoglobin Electrophoresis, Molecular

81257 x 2-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)

81404-HBB (hemoglobin, beta, Beta-Globin) (eg, thalassemia), full gene sequence (if appropriate)

Alpha Globin Gene Analysis

83891-Isolation or extraction of highly purified nucleic acid (if appropriate)

83894-Separation by gel electrophoresis (if appropriate)

83900 x 2-Amplification, target, multiplex, first 2 nucleic acid sequences (if appropriate)

83909-Separation and identification by high-resolution technique (if appropriate)

83912-Interpretation and report (if appropriate)

83914 x15-Mutation identification by enzymatic ligation or primer extension, single segment, each segment (if appropriate)

Hemoglobin S, Screen, Blood

85660 (if appropriate)

Hemoglobin F, Red Cell Distribution, Blood

88184 (if appropriate)