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Epic Code LAB1230788 Thalassemia and Hemoglobinopathy Evaluation, Blood and Serum

Additional Codes

Mayo code: THEV1

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Useful For

Evaluation of microcytosis

 

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

 

Diagnosis of hereditary persistence of hemoglobin

Specimen Type

Serum
Whole Blood EDTA


Necessary Information


Include recent transfusion information.

 

Include most recent complete blood cell count results.

 

Include ferritin results if not sending serum

 

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.



Specimen Required


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

Container/Tube:

Preferred: Serum gel

Acceptable: Red top

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.


Laboratory Test Directory Note:

COLLECTION NOTE: Volumes listed are in serum or plasma, draw approximately 2 1/2 times the requested volume in whole blood.

Specimen Minimum Volume

Blood: 2.5 mL
Serum: 0.5 mL

Forms

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 in Special Instructions:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing-Spanish (T826)

2. Metabolic Hematology Patient Information (T810) in Special Instructions

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

Specimen Stability Information

Specimen Type Temperature Time Special Container
Serum Refrigerated 7 days
Whole Blood EDTA Refrigerated 7 days

Reject Due To

Gross hemolysis Reject

Cautions

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

Clinical Information

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.

Method Name

THEVI, THEV0: Medical Interpretation

HGBCE: Capillary Electrophoresis

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

FERR: Immunoenzymatic Assay

IEF: Isoelectric Focusing

MASS: Mass Spectrometry (MS)

HPFH: Flow Cytometry

UNHB: Isopropanol and Heat Stability

Report Available

2 to 25 days

Reporting Name

Thalassemia and Hemoglobinopathy Ev

Reference Values

Definitive results and an interpretive report will be provided.

Interpretation

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

Method Description

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; instruction manual: Bio-Rad Variant II Beta-thalassemia Short Program Instructions for Use, L70203705. Bio-Rad Laboratories, Inc; 11/2011)

 

Ferritin:

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. A chemiluminescent substrate 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: Access Ferritin. Beckman Coulter, Inc; 11/2019)

Day(s) Performed

Monday through Thursday

Clinical Reference

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;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. 2013;36:1-12

Test Classification

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

CPT Code Information

83020-26-Hemoglobinopathy Interpretation

83020-Hb Variant, A2 and F Quantitation

83021-HPLC Hb Variant

82728-Ferritin

82664 (if appropriate)

83068 (if appropriate)

83789 (if appropriate)

88184 (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
THEV1 Thalassemia and Hemoglobinopathy Ev In Process

 

Result ID Test Result Name Result LOINC Value
FERR Ferritin, S 20567-4
41927 Hb A 20572-4
65615 HPLC Hb Variant, B No LOINC Needed
608425 Hemoglobinopathy Interpretation 14869-2
608868 Reviewed By 18771-6
41928 Hb F 4576-5
41929 Hb A2 4551-8
41930 Variant 1 24469-9
41931 Variant 2 24469-9
41932 Variant 3 24469-9
41933 HGBCE Interpretation 78748-1

Profile Information

Test ID Reporting Name Available Separately Always Performed
THEVI Hemoglobinopathy Interpretation No Yes
HGBCE Hb Variant, A2 and F Quantitation,B Yes Yes
HPLC HPLC Hb Variant, B No Yes
FERR Ferritin, S Yes Yes

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
HPFH Hb F Distribution, B No No
SDEX Sickle Solubility, B Yes No
IEF Isoelectric Focusing, B No No
UNHB Hb Stability, B No No
MASS Hb Variant by Mass Spec, B No No
ATHAL Alpha-Globin Gene Analysis Yes No
WASQR Alpha Globin Gene Sequencing, B Yes, (Order WASEQ) No
WBSQR Beta Globin Gene Sequencing, B Yes, (Order WBSEQ) No
WBDDR Beta Globin Cluster Locus Del/Dup,B Yes, (Order WBDD) No
WGSQR Gamma Globin Full Gene Sequencing Yes, (Order WGSEQ) No
THEV0 Thalassemia Summary Interpretation No No