Test Id : THEV1

This is a consultative evaluation in which the case will be evaluated at Mayo Clinic Laboratories, the appropriate tests performed at an additional charge, and the results interpreted.

This evaluation will always include hemoglobins A2 and F and hemoglobin electrophoresis utilizing cation exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis methods.

If a serum sample is received, a serum ferritin will always be performed to allow incorporation of possible iron deficiency into profile interpretation and economical test utilization. If the ferritin component is not needed, do not send a serum sample, and the ferritin test will not be performed. Note: If a ferritin is not performed or provided, and if microcytosis is present and no other abnormalities are found (beta thalassemia, a hemoglobin variant that is associated with microcytosis), the case will be reflexed to alpha-globin gene analysis unless otherwise requested not to be performed.

Hemoglobin electrophoresis reflex testing, performed at additional charge, may include any or all of the following as indicated to identify rare hemoglobin variants present: sickle solubility (hemoglobin S screen), hemoglobin heat and isopropanol stability studies, isoelectric focusing, HbF distribution by flow cytometry, cation exchange HPLC, DNA (Sanger) testing for beta-chain variants and the most common beta thalassemias (beta-globin gene sequencing), multiplex ligation-dependent probe amplification testing for beta-cluster locus large deletions and duplications, including large deletional hereditary persistence of fetal hemoglobin (HPFH), delta-beta, delta thalassemias, gamma-delta-beta, and epsilon-gamma-delta-beta thalassemias (beta-globin cluster locus deletion/duplication), large deletional alpha thalassemias and alpha-gene duplications (alpha-globin gene analysis), alpha-chain variants and nondeletional alpha thalassemias (alpha-globin gene sequencing), and gamma-chain variants and nondeletional HPFH (gamma-globin full gene sequencing).

An additional consultative interpretation that summarizes all testing will be provided after test completion to incorporate subsequent results into overall evaluation if any of the following molecular tests are reflexed on this test.

-ATHAL / Alpha-Globin Gene Analysis, Varies

-WASQR / Alpha-Globin Gene Sequencing, Blood

-WBSQR / Beta-Globin Gene Sequencing, Blood

-WBDDR / Beta-Globin Cluster Locus Deletion/Duplication, Blood

-WGSQR / Gamma-Globin Full Gene Sequencing, Varies

The results of the individual protein and molecular tests will be released as they are completed; with a final summary interpretation report correlating all performed testing with any clinical information or complete blood cell count results received.

For more information see Benign Hematology Evaluation Comparison

Include the following information with the specimen:

Recent transfusion information

-Most recent complete blood cell count results

-If not sending serum, include ferritin results.

Metabolic Hematology Patient Information (T810) is strongly recommended. Testing may proceed without this information, however if the information requested is received, any pertinent reported clinical features and data will drive the focus of the evaluation and be considered in the interpretation.

The laboratory has extensive experience in hemoglobin variant identification and many cases can be confidently classified without molecular testing. However, molecular confirmation is always available, subject to sufficient sample quantity (eg, MLPA testing requires at least 2 mL of sample in addition to protein testing requirements). If no molecular testing or specific molecular tests are desired, utilize the appropriate check boxes on the form. If the form or other communication is not received, the reviewing hematopathologist will select appropriate tests to sufficiently explain the protein findings which may or may not include molecular testing.

Blood and serum are required.

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 15 mL

Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.

Specimen Type: Serum

Patient Preparation: For 12 hours before specimen collection do not take multivitamins or dietary supplements containing biotin (vitamin B7), which is commonly found in hair, skin, and nail supplements and multivitamins.

Collection Container/Tube:

Preferred: Serum gel

Acceptable: Red top

Submission Container/Tube: Plastic vial

Specimen Volume: 0.6 mL

Collection Instructions:

1. Serum gel tubes should be centrifuged within 2 hours of collection.

2. Red-top tubes should be centrifuged, and the serum aliquoted into a plastic vial within 2 hours of collection.

3. Label specimen as serum.

• Informed Consent for Genetic Testing
• Metabolic Hematology Patient Information
• Benign Hematology Evaluation Comparison
• Informed Consent for Genetic Testing (Spanish)

1. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing-Spanish (T826)

2. Metabolic Hematology Patient Information (T810)

3. If not ordering electronically, complete, print, and send a Benign Hematology Test Request (T755) with the specimen

Blood: 2.5 mL

Serum: 0.5 mL

Evaluation of microcytosis

Extensive and economical diagnosis and classification of hemoglobinopathies or thalassemia, including complex disorders

Diagnosis of hereditary persistence of hemoglobin

This is a consultative evaluation in which the case will be evaluated at Mayo Clinic Laboratories, the appropriate tests performed at an additional charge, and the results interpreted.

This evaluation will always include hemoglobins A2 and F and hemoglobin electrophoresis utilizing cation exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis methods.

If a serum sample is received, a serum ferritin will always be performed to allow incorporation of possible iron deficiency into profile interpretation and economical test utilization. If the ferritin component is not needed, do not send a serum sample, and the ferritin test will not be performed. Note: If a ferritin is not performed or provided, and if microcytosis is present and no other abnormalities are found (beta thalassemia, a hemoglobin variant that is associated with microcytosis), the case will be reflexed to alpha-globin gene analysis unless otherwise requested not to be performed.

Hemoglobin electrophoresis reflex testing, performed at additional charge, may include any or all of the following as indicated to identify rare hemoglobin variants present: sickle solubility (hemoglobin S screen), hemoglobin heat and isopropanol stability studies, isoelectric focusing, HbF distribution by flow cytometry, cation exchange HPLC, DNA (Sanger) testing for beta-chain variants and the most common beta thalassemias (beta-globin gene sequencing), multiplex ligation-dependent probe amplification testing for beta-cluster locus large deletions and duplications, including large deletional hereditary persistence of fetal hemoglobin (HPFH), delta-beta, delta thalassemias, gamma-delta-beta, and epsilon-gamma-delta-beta thalassemias (beta-globin cluster locus deletion/duplication), large deletional alpha thalassemias and alpha-gene duplications (alpha-globin gene analysis), alpha-chain variants and nondeletional alpha thalassemias (alpha-globin gene sequencing), and gamma-chain variants and nondeletional HPFH (gamma-globin full gene sequencing).

An additional consultative interpretation that summarizes all testing will be provided after test completion to incorporate subsequent results into overall evaluation if any of the following molecular tests are reflexed on this test.

-ATHAL / Alpha-Globin Gene Analysis, Varies

-WASQR / Alpha-Globin Gene Sequencing, Blood

-WBSQR / Beta-Globin Gene Sequencing, Blood

-WBDDR / Beta-Globin Cluster Locus Deletion/Duplication, Blood

-WGSQR / Gamma-Globin Full Gene Sequencing, Varies

The results of the individual protein and molecular tests will be released as they are completed; with a final summary interpretation report correlating all performed testing with any clinical information or complete blood cell count results received.

For more information see Benign Hematology Evaluation Comparison

This consultative study is primarily designed for the evaluation of microcytosis but also has the ability to test for the detection of almost all known hemoglobin disorders in an economical manner. Because this can include multiple tests for alpha thalassemias, beta thalassemias, delta-beta thalassemia, hereditary persistence of fetal hemoglobin (HPFH), and for all known hemoglobin (Hb) variants, an expert in these disorders can guide testing to explain the clinical question or reported complete blood cell count values. This evaluation is particularly useful for complete classification of compound combinations of HbS with alpha or beta thalassemia, HbE/beta-0-thalassemia, and many other complex alpha and beta thalassemia disorders. Since iron deficiency can mimic thalassemias, ferritin levels are measured to evaluate this possibility if a serum sample is received.

Hb disorders include those associated with thalassemias (decreased protein quantity) and Hb variants (abnormal protein production). Many are clinically harmless, and others cause symptoms, including microcytosis, sickling disorders, hemolysis, erythrocytosis, cyanosis/hypoxia, long-standing or familial anemia, compensated or episodic anemia, and increased methemoglobin or sulfhemoglobin results. Hb disorders can show patterns of either autosomal recessive or autosomal dominant inheritance.

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

The most common form of thalassemia is alpha thalassemia. Alpha thalassemia usually involves deletion of entire alpha genes and varies in severity depending on the number of alpha chains deleted (or rendered nonfunctional). Alpha thalassemia trait usually results from the deletion of 2 alpha genes. The most common form of HbH disease, results from dysfunction of 3 alpha chains, and shows a variable phenotype with most showing moderate anemia. The deletion of all 4 alpha genes (Barts hydrops fetalis) is incompatible with life without significant medical intervention. Nondeletional alpha thalassemia alterations can also result in either thalassemia trait or HbH disease and are less common than deletional forms.

Conversely most beta thalassemia alterations are due to single nucleotide substitutions that can occur anywhere in the beta-globin gene. Large deletions of the beta-globin gene complex can result in elevations in HbF, such as HPFH or delta-beta thalassemia. While the presence of a single beta-gene variants (beta thalassemia trait) results primarily in red blood cells microcytosis, cases with 2 beta-gene abnormalities show a wide range in clinical severity, and many cases require molecular testing to understand the phenotype.

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 alterations that include most, but not all, alpha thalassemias.

1. Hoyer JD, Hoffman DR: The thalassemia and hemoglobinopathy syndromes. In: McClatchey KD, Amin HM, Curry JL, eds. Clinical Laboratory Medicine. 2nd ed. Lippencott Williams and Wilkins; 2002:866-892

2. Brancaleoni V, Di Pierro E, Motta I, Cappellini MD: Laboratory diagnosis of thalassemia. Int J Lab Hematol. 2016 May;38 Suppl 1:32-40

3. Hartveld C: State of the art and new developments in molecular diagnostics for hemoglobinopathies in multiethnic societies. Int J Lab Hematol. 2014 Feb;36:1-12

Hemoglobin 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 hemolyzing 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 electropherogram 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: HbA2', C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart, J, N-Baltimore, and H.(Louahabi A, Philippe M, Lali S, Wallemacq P, Maisin D: 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; Instruction manual: CAPILLARYS Hemoglobin(E) using the CAPILLARYS 2 flex-piercing instrument. Sebia; 06/2014)

High-Performance Liquid Chromatography Hemoglobin Variant:

Hemolysate of whole blood is injected into an analysis stream passing through a cation exchange column using high-performance liquid chromatography. 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 absorbance are displayed as a chromatogram of absorbance versus time.( Huismann TH, Scroeder WA, Brodie AN, Mayson SM, Jakway J: 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; Szuberski J, Oliveira JL, Hoyer JD: A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC). Int J Lab Hematol. 2012 Dec; 34(6):594-604; Instruction manual: Bio-Rad Variant II Beta-thalassemia Short Program Instructions for Use, L70203705. Bio-Rad Laboratories, Inc; 11/2011)

Ferritin:

The Roche ferritin method employs monoclonal antibodies specifically directed against ferritin. A biotinylated monoclonal antibody and a second monoclonal antibody labeled with a ruthenium complex react to form a sandwich complex. After the addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Application of a voltage to the electrode then induces chemiluminescent emission, which is measured by a photo multiplier. (Package insert: Elecsys Ferritin. Roche Diagnostics; 10/2022)

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

83020-26-Hemoglobinopathy Interpretation

83020-Hb Variant, A2 and F Quantitation

83021

82728

82664 (if appropriate)

83068 (if appropriate)

83789 (if appropriate)

88184 (if appropriate)