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Test Code HBELB Hemoglobin Electrophoresis Evaluation, Blood


Necessary Information


Include recent transfusion information.

 

Include most recent complete blood cell count 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, multiplex ligation-dependent probe amplification testing requires at least 2 mLs 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


Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: ACD (solution B), green top (sodium heparin)

Specimen Volume: 10 mL

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


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

Secondary ID

608083

Useful For

Diagnosis and classification of hemoglobin disorders, including thalassemias and hemoglobin variants

Profile Information

Test ID Reporting Name Available Separately Always Performed
HBELI Hb Electrophoresis Interpretation No Yes
HGBCE Hb Variant, A2 and F Quantitation,B Yes Yes
HPLC HPLC Hb Variant, B No Yes

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
HPFH Hb F Distribution, B No No
MASS Hb Variant by Mass Spec, B No No
SDEX Sickle Solubility, B Yes No
IEF Isoelectric Focusing, B No No
UNHB Hb Stability, 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 No No
HBEL0 Hb Electrophoresis Summary Interp No No

Testing Algorithm

Hemoglobin electrophoresis evaluation will always include hemoglobin A(2) and F and hemoglobin electrophoresis utilizing capillary electrophoresis (CE) and cation exchange high-performance liquid chromatography (HPLC) methods.

 

Hemoglobin electrophoresis reflex testing, performed at additional charge, may include any or all of the following to identify rare hemoglobin variants present: sickle solubility (hemoglobin S screen); hemoglobin heat and isopropanol stability studies (unstable hemoglobin); isoelectric focusing (IEF), intact globin chain mass spectrometry (hemoglobin variant by mass spectrometry); Hb F distribution by flow cytometry (hemoglobin F distribution); DNA (Sanger) sequencing assays for: 1) beta chain variants and the most common beta thalassemias (beta globin gene sequencing), 2) alpha chain variants and less common non-deletional alpha thalassemias (alpha globin gene sequencing), or 3) gamma chain variants and non-deletional hereditary persistence of fetal hemoglobin (HPFH) (gamma globin full gene sequencing); multiplex ligation-dependent probe amplification (MLPA) assays for: 1) large deletional alpha thalassemias and alpha gene duplications (alpha-globin gene analysis), or 2) beta cluster locus large deletions and duplications, including large deletional HPFH, delta-beta thalassemia (DBT), gamma-delta-beta thalassemia (GDBT), epsilon-gamma-delta-beta thalassemia (EGDBT) and large deletional delta thalassemia. (beta globin cluster locus del/dup),

 

If test results in the profile are abnormal, results may be reviewed by a hematopathology consultant and a summary interpretation provided.

 

One or more of the following molecular tests may be reflexed:

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;

 

For cases with molecular testing added, a preliminary interpretation will be reported that discusses the protein test results. After all test results are finalized, an additional consultative interpretation that summarizes all testing and incorporates subsequent genetic results will be provided.

 

See Benign Hematology Evaluation Comparison in Special Instructions.

Method Name

HBELI: Consultative Interpretation

HGBCE: Capillary Electrophoresis (CE)

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

IEF: Isoelectric Focusing (IEF)

MASS: Mass Spectrometry (MS)

HPFH: Flow Cytometry

UNHB: Isopropanol and Heat Stability

HBEL0: Medical Interpretation

Reporting Name

Hb Electrophoresis Evaluation

Specimen Type

Whole Blood EDTA

Specimen Minimum Volume

1 mL (this volume will limit reflex testing possibilities)
3 mL if multiplex ligation-dependent probe amplification is desired

Specimen Stability Information

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

Reject Due To

All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.

Reference Values

HEMOGLOBIN A

1-30 days: 5.9-77.2%

1-2 months: 7.9-92.4%

3-5 months: 54.7-97.1%

6-8 months: 80.0-98.0%

9-12 months: 86.2-98.0%

13-17 months: 88.8-98.0%

18-23 months: 90.4-98.0%

≥24 months: 95.8-98.0%

 

HEMOGLOBIN A2

1-30 days: 0.0-2.1%

1-2 months: 0.0-2.6%

3-5 months: 1.3-3.1%

≥6 months: 2.0-3.3%

 

HEMOGLOBIN F

1-30 days: 22.8-92.0%

1-2 months: 7.6-89.8%

3-5 months: 1.6-42.2%

6-8 months: 0.0-16.7%

9-12 months: 0.0-10.5%

13-17 months: 0.0-7.9%

18-23 months: 0.0-6.3%

≥24 months: 0.0-0.9%

 

VARIANT 1

0.0

 

VARIANT 2

0.0

 

VARIANT 3

0.0

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. Lippincott Williams and Wilkins; 2002: 866-895

2. Oliveira JL: Diagnostic strategies in hemoglobinopathy testing, the role of a reference laboratory in the USA. Thalassemia Reports, 2018; 8(1). doi: 10.4081/thal.2018.7476

3. Brancaleonai V, Di Pierro E, Motta I, Cappellini MD: Laboratory diagnosis of thalassemia. Int J Lab Haematol. 2016; 38(suppl 1):32-40

4. Hartveld CI: State of the art and new developments in molecular diagnostics for hemoglobinopathies in multiethnic societies. Int J Lab Haematol. 2014; 36:1-12

5. 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

6. Riou J, Szuberski J, Godart C, Wajcman H, Oliveira JL, Hoyer JD, Bardakdjian-Michau J. Precision of CAPILLARYS 2 for the detection of hemoglobin variants based on their migration positions. Am J Clin Pathol. 2018 Jan 29;149 (2):172-180

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 hemoglobin. The resulting electrophoregram peaks are evaluated for pattern abnormalities and are quantified as a percentage of the total hemoglobins 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; instruction manual: CAPILLARYS Hemoglobin(E) using the CAPILLARYS 2 flex-piercing instrument. Sebia; 06/2014)

 

High-Performance Liquid Chromatography:

Hemolysate of whole blood is injected into an analysis stream passing through a cation exchange column using high-performance liquid chromatography (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 absorbance are displayed as a chromatogram of absorbance 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; instruction manual: Bio-Rad Variant II Beta-thalassemia Short Program Instructions for Use, L70203705. Bio-Rad Laboratories, Inc; 11/2011)

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test has been modified from the manufacturer's instructions. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.

CPT Code Information

83020-Quantitation by electrophoresis

83021-Quantitation by HPLC

82664-Electrophoresis, not elsewhere specified (if appropriate)

83068 (if appropriate)

83789 (if appropriate)

88184 (if appropriate)

83020-26 (if appropriate)

 

LOINC Code Information

Test ID Test Order Name Order LOINC Value
HBEL1 Hb Electrophoresis Evaluation 43113-0

 

Result ID Test Result Name Result LOINC Value
41927 Hb A 20572-4
65615 HPLC Hb Variant, B No LOINC Needed
608088 Hb Electrophoresis Interpretation 49316-3
609421 Hb Electrophoresis Interp Cancel No LOINC Needed
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